CN102268432A - Orotate phosphoribosyltransferase promoter, application, construct and vector - Google Patents

Abstract

With the present invention, upstream sequence and downstream sequence of orotate phosphoribosyltransferase genomic DNA of rhodosporidium toruloides are amplified, biological information analysis and functional verification are performed for the amplified sequences to obtain the promoter and the terminator, wherein a target gene can be effectively expressed in the rhodosporidium toruloides through the promoter and terminator, such that the promoter and terminator can be applicable for the rhodosporidium toruloides genetic engineering operation and the strain improvement. The invention further relates to the DNA construct containing the components and the vector containing the components.

Description

Orotate phosphoribosyl transferase promotor and application and construct and carrier

Technical field

This patent belongs to gene engineering technology field, is specifically related to the red winter spore Yeast promoter of circle, terminator and uses thereof, comprises the necessary method for transformation of construction method of gene engineering strain etc.

Background technology

Microorganism is occurring in nature distribution one of species the most widely, has remarkable biosynthesis ability, almost can synthesize organic chemicals all on the earth.Kind diversity and the genetic diversity of microorganism have determined its metabolism diversity.Compare with multicellular organism, though the pathways metabolism of microorganism is simple relatively, the production of its compound is efficient, quick, and in close relations with the daily productive life of the mankind.

Bacterial strain is used in natural production bacterial strain or environmental improvement as a certain chemical, and its specific production performance often is not an optimization.How optimizing or change the metabolism network and the expression regulation network of industrial strain, with accumulating rate or the directed quality of controlling the target product that improves the bio-based product, is the focus and the difficult point of current biological technical field research.Whether in fact, can reach or surpass the chemical process level understanding, the development and utilization of microbial oil metabolic process, also be the key that improves the fermenting process economy.The progress of genome sequencing and genetic engineering technique is for the understanding of bacterial strain physiology characteristic and strain improvement provide than traditional induced-mutation technique reasonable method more.

The essence of metabolic engineering is to utilize recombinant DNA technology and other technology, on purpose changes existing metabolism and expression regulation network, understands and utilize the pathways metabolism of cell better.Metabolic engineering can be familiar with and the engineered cells process on cell and molecular level, and it not only can explain the cell physiological biochemical characteristic, but also can give new proterties of starting strain and phenotype: (1) enlarges the substrate utilization scope; (2) produce original non-existent new compound; (3) enhancing is to the degradation capability of harmful toxic matter in the environment; (4) improve the adaptive faculty of thalline to environment; (5) generation of blocking-up or reduction byproduct; (6) (Bailey J E.Toward a science ofmetabolic engineering.Science.1991,252 (5013): 1668-1675 such as raising of metabolism products production speed and production performance; Aristidou A,

M.Metabolic engineering applications to renewable resource utilization.Curr.Opin.Biotechnol.2000,11 (2): 187-198).

The circle Rhodosporidium is in Basidiomycota heterothally type fungi, it is a kind of very important microorganism in the fermentation industry, can utilize the hexose and the pentose that come from biomass to be the important bio-based product of raw material production: microbial oil, grease can reach (the Ratledge C more than 60% of dry cell weight in the born of the same parents, WynnJ P.The biochemistry and molecular biology of lipid accumulation in oleaginousmicroorganisms.Adv.Appl.Microbiol.2002,51:1-51; Li Y, Zhao Z, Bai F.High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4infed-batch culture.Enzyme Microb.Technol.2007,41 (3): 312-317); Industrial enzymes or medicine synthetic usefulness enzyme such as phosphodiesterase, phenylalanine ammonia lyase (Hodgins D S.Yeastphenylalanine ammonia-lyase.Purification, properties, and the identification ofcatalytically essential dehydroalanine.J Biol.Chem.1971,246 (9): 2977-2985; Gilbert H J, Clarke I N, Gibson R K, et al.Molecular cloning of the phenylalanineammonia lyase gene from Rhodosporidium toruloides in Escherichia coli K-12.JBacteriol.1985,161 (1): 314-320), D amino-acid oxidase (Gadda G Negri A, PiloneM S.Reaction of phenylglyoxal with arginine groups in D-amino-acid oxidasefrom Rhodotorula gracilis.J Biol.Chem.1994,269 (27): 17809-17814; Liao G J, Lee Y J, Lee Y H, et al.Structure and expression of the D-amino-acid oxidasegene from the yeast Rhodosporidium toruloides.Biotechnol.Appl.Biochem.1998,27 (Pt 1): 55-61) etc.; β-Hu Luobusu and exocellular polysaccharide; And in sewage disposal and bio-pharmaceuticals, have more widely and to use.Experimental result shows, this bacterium can utilize five-carbon sugar and hexose to be substrate simultaneously, good stress resistance, can be carbon source accumulation grease directly, can realize efficient conversion (Li Yonghong, the Liu Bo of biomass to the bio-based product with the maize straw acid hydrolysis liquid, Sun Yan, Deng. the screening of wide spectrum carbon source oleaginous yeast bacterium. Chinese biological engineering magazine, 2005,25 (12): 39-44).

Though the red winter spore yeast of circle has good industrial production performance, simultaneously, the proteolytic enzyme heterogenous expression in circle red winter spore yeast source (the Pollegioni L that also succeeds, Molla G, Campaner S, Cloning, sequencing and expression in E.coli of a D-amino acid oxidase cDNA fromRhodotorula gracilis active on cephalosporin C.J Biotechnol.1997,58 (2): 115-123; Faulkner J D, Anson J G, Tuite M F, et al.High-level expression of thephenylalanine ammonia lyase-encoding gene from Rhodosporidium toruloidesin Saccharomyces cerevisiae and Escherichia coli using a bifunctionalexpression system.Gene.1994,143 (1): 13-20), but the red winter spore yeast of circle self lacks corresponding genetic operating system.With the red winter spore yeast of circle is the host bacterium, no matter is genetically engineered operation or the improvement of metabolic engineering bacterial strain, all is subjected to the restriction of genetic operating system, is difficult to carry out the bacterial strain transformation of target.

Promotor is essential for genetic operating system.Therefore, if the red winter spore yeast of circle is carried out genetic manipulation, the promotor that acquisition can be worked in the red winter spore yeast of circle is the key link of this work.

Summary of the invention

In view of above-mentioned prior art bottleneck, main purpose of the present invention provides and is used in effective expression foreign gene in the red winter spore yeast of circle, and can be used for justifying by gene engineering the promotor and the terminator of red winter spore barms improvement.

For realizing purpose of the present invention, the inventor furthers investigate the expression conditions in the red winter spore yeast of circle, finds that ura5 is a constitutive expression gene, and its promotor is middle strong promoter.Design by experiment in conjunction with methods such as degenerate pcr, chromosome walkings, has successfully obtained to comprise the dna fragmentation of effective promotor from the red winter spore yeast chromosomal dna of circle, finished the present invention thus.

Specifically, the present invention comprises following embodiment (A) to (H)

(A) the present invention relates to a kind of active dna fragmentation of the red winter spore yeast transcripting promoter of circle that has, described dna fragmentation have the full sequence of dna sequence dna shown in SEQ ID NO:1 or comprise this dna sequence dna from 3 '-terminal 800bp is with interior partial sequence, or have and to play 800bp with interior partial sequence hybridization with whole or its dna sequence dna 3 '-end of sequence shown in SEQ ID NO:1, and keep the active sequence of transcripting promoter, or the deoxynucleoside acid sequence shown in the SEQ ID NO:1 carried out the replacement of one or more bases, disappearance, insertion or interpolation are obtained, and have 50% above homology with sequence shown in the SEQ ID NO:1, and sequence with promoter activity.

(B) the present invention relates to a kind of red winter spore zymic dna fragmentation of justifying by oneself, described dna fragmentation has following feature: (1) dna sequence dna shown in SEQ ID NO:2 whole or comprise the partial sequence of this dna sequence dna 5 '-end; (2) or have can with sequence hybridization shown in (1) and keep as the active sequence of sequence as described in (1).

(C) the present invention relates to a kind of target gene is transcribed the initial sum Transcription Termination in the red winter spore yeast of circle dna molecular of finishing, it has simultaneously as sequence as described in (A) with as sequence as described in (B), and be positioned at downstream as sequence as described in (B), be adjacent the dna fragmentation of 1-10000 Nucleotide as sequence as described in (A).

(D) the present invention relates to a kind of DNA construct that target gene can be connected with (A)-(C) described any dna molecular, so that the recombinant DNA that target gene can be expressed in the red winter spore yeast of circle.Described target gene is encoding histone nucleic acid or antisense nucleic acid coding nucleic acid.

(E) the present invention relates to any one carrier in the described dna molecular of a kind of carrying (A)-(D).Described carrier can be plasmid vector or cosmid vector.

(F) the present invention relates to have changed over to as (D) described dna molecular or as the spore yeast of red winter of circle or Rhodosporidium (Rhodosporidium) fungi of carrier as described in (E).

(G) involved in the present invention pRtura5 promotor, it is characterized in that: it derives from the red winter spore yeast of circle, can start the transcript and expression of goal gene in the red winter spore yeast of circle.

(H) the present invention relates to the dna fragmentation that coding has the active polypeptide of orotate phosphoribosyl transferase (orotatephosphoribosyl transferase), its cDNA sequence has the deoxynucleoside acid sequence shown in SEQ ID NO:3, its genomic dna has the deoxynucleoside acid sequence shown in the SEQ ID NO:5, and its aminoacid sequence is shown in SEQ ID NO:4.

Use and have the dna molecular of promoter activity among the present invention and have the active DNA of transcription terminator, can realize foreign gene or native gene in the expression of circle in the red winter spore yeast, the invention provides and be used for genetic engineering circle red winter spore zymic promotor, terminator and carrier.For the red winter spore yeast of circle has been opened a breeding new way, and therefore can provide the spore yeast of the red winter of novel circle with industrial use.

Description of drawings

Fig. 1 represents the result of the agarose gel electrophoresis of Rtura5 degenerate pcr product.

Fig. 2 represents the result of the agarose gel electrophoresis of pRtura5 promoter dna fragment.

Fig. 3 represents the result of the agarose gel electrophoresis of Rtura5t terminator dna fragmentation.

Fig. 4 represents the result of the agarose gel electrophoresis of Rtura5 promotor-ORF-terminator full length fragment.

Fig. 5 represents that the pRtura5 promotor starts the conformability expression of results of GFPuv in the red winter spore yeast ATCC 10788 of circle.

Fig. 6 represents that the pRtura5 promotor starts the antibiosis expression of results of bleomycin resistant gene ble at red winter spore yeast ATCC10788.

Fig. 7 represents that the pRtura5 promotor starts the antibiosis expression of results of Geneticin resistant gene kanmx4 at red winter spore yeast ATCC 10788.

Fig. 8 represents the cultivation results of the reorganization red winter spore yeast of circle on 5 '-FOA.

Fig. 9 is the structure iron of plasmid pura5gfp.

Figure 10 is the structure iron of plasmid pura5ble.

Figure 11 is the structure iron of plasmid pura5kan.

Figure 12 is the crystal structure model of the red winter spore yeast orotate phosphoribosyl transferase OPRTase of circle, and it shows that three-dimensional structure is correct.

The embodiment that this is bright

In this article, " promotor " be meant can be discerned by RNA polymerase, in conjunction with and the dna sequence dna of can promotor gene transcribing.Term " promotor " also can be regarded as: comprise 5 ' non-coding region, cis-acting elements (as enhanser) and other can with transcription factor bonded nucleotide sequence.

The existence or the intensity of promotor are normally represented by promoter activity, its measuring method: the downstream of promotor as described in reporter gene (as green fluorescent protein) is connected in, and this DNA construct transformed corresponding host cell, the examining report gene expression amount.If people observe the expression that is connected in described promotor downstream reporter gene, just can think that described promotor has activity in its institute's transformed host cells.

In this article, " terminator " is meant and provides termination signal that RNA polymerase is separated with dna profiling on the karyomit(e) and make the section of DNA sequence of Transcription Termination.Can confirm the existence of terminator by check the size of institute's transcribe rna such as the method for Northern hybridization or RT-PCR.Or make the reporter gene effective expression determine the activity of terminator by " promotor-reporter gene-terminator " construct.

" the red winter spore yeast of circle " among the present invention comprises any diploid and the monoploid, wild type strain and the auxotrophic strain that belong to these " species "." Rhodosporidium fungi " among the present invention is not specifically limited, and its example comprises the fungi that belongs to this genus, except that the red winter spore yeast of circle, as Rhodosporidium azoricum, Rhodosporidium babjevae, Rhodosporidiumsphaerocarpum.

" goal gene " of the present invention, comprise can the circle red winter spore yeast in expressed proteins encoding sequence, sense-rna encoding sequence and nuclease encoding sequence.Can be in the red winter spore yeast of circle the example of expressed proteins encoding sequence comprise and come from the red winter spore zymic nucleotide sequence of circle, and be not limited thereto.Goal gene of the present invention also comprises the albumen coded sequence that derives from other microorganism, plant and animal.

Promotor among the present invention has the whole of shown in SEQ ID NO:1 dna sequence dna or comprises the partial sequence of this dna sequence dna 3 '-end, or have can with the partial sequence hybridization of whole or its dna sequence dna 3 '-end of sequence shown in SEQ ID NO:1 and keep the active sequence of transcripting promoter, or to the deoxynucleoside acid sequence shown in the SEQ ID NO:1 carry out one or more bases replacement, disappearance, insertion or interpolation obtained, have 50% above homology and have the sequence of promoter activity with sequence shown in the SEQ ID NO:1.

Terminator among the present invention has as the whole of the dna sequence dna shown in SEQ ID NO:2 or comprises the partial sequence of this dna sequence dna 5 '-end, or have can with the partial sequence hybridization of whole or its dna sequence dna 5 '-end of sequence shown in SEQ ID NO:2 and keep the active sequence of transcription terminator, or to the deoxynucleoside acid sequence shown in the SEQ ID NO:2 carry out one or more bases replacement, disappearance, insertion or interpolation obtained, have 50% above homology and have the active sequence of terminator with sequence shown in the SEQ ID NO:2.

The construct of the construct of the promotor-goal gene among the present invention, the construct of goal gene-terminator or promotor-goal gene-terminator, can directly or through carrier mediated conversion justify red winter spore yeast, so that destination gene expression, can the preferred plasmid carrier as the mediation carrier.

Promotor of the present invention can be separated from the red winter spore yeast of circle according to following aspect.

The invention will be further described below in conjunction with drawings and Examples, will help those of ordinary skill in the art to understand the present invention, but not limit the present invention in any form.Synthetic and the examining order of all primers is then finished by Dalian TaKaRa company if no special instructions among the following embodiment.

Embodiment 1: the extraction of the red winter spore yeast ATCC 10788 total RNA of circle

With spore yeast R.toruloides of red winter of fresh circle ATCC 10788 (available from the biological product of USS collecting center, ATCC) be inoculated in the 50ml YEPD liquid nutrient medium by the inclined-plane, cultivate 24h in 30 ℃ of shaking tables, with 1: 50 volume ratio bacterium liquid is transferred to respectively in the 100ml YEPD liquid nutrient medium again, cultivates 12h in 30 ℃ of shaking tables and reach logarithmic phase.Under 4 ℃, the centrifugal 4min of 5000rpm collects thalline, with the rapid freezing thalline of liquid nitrogen, grinds broken wall.Use the TaKaRa RNAiso of company test kit, and extract total RNA according to its standard step.

RNA carries out 1.5% agarose gel electrophoresis, uses fluorescence-uv analyzer to observe and identifies, as seen two bands clearly.With the total RNA sample of ultraviolet spectrometer analysis, record OD ₂₆₀/ OD ₂₈₀=2.01, show that total RNA quality is fine.Total RNA sample is frozen in-80 ℃, standby.

Embodiment 2: the synthetic and ura5 degenerate pcr of red winter spore yeast ATCC 10788 cDNA first chain of circle

Red winter spore yeast R.toruloides ATCC 10788 total RNA are template with circle, and cDNA first chain is synthesized in reverse transcription.At first, with the total RNA of 1.0 μ l (about 2 μ g), 1.0 μ l primer SMART IV:5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3 ' and 1.0 μ loligo dT-joint primer CDS III/3 ': 5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) _30N-1N-3 ', 2.0 μ l DEPC treating water (diethylpyrocarbonate treating water, available from Dalian TaKaRa company), join mixing in the PCR pipe, in 72 ℃ of insulation 2min, place cooled on ice 2min immediately, with 2.0 μ l, 5 * first strandbuffer, 1.0 μ l DTT (20mM), 1.0 μ l dNTP (10mM), 1.0 μ l powerscript reversetranscriptase (Clontech company) joins in the system mixing.In 42 ℃ of extension 60min, last 4 ℃ are finished reaction, are stored in-20 ℃, standby.

The orotate phosphoribosyl transferase aminoacid sequence of other source of species of announcing according to NCBI, obtain its relative conservative region by sequence alignment, and utilize this conservative region design to synthesize two degenerated primers, ura5-sense:5 '-ATGAG (AGCT) GC (AGCT) AC (AGCT) TCCTA (AGCT) GC-3 ' and ura5-anti:5 '-CTA (AGCT) T (CT) be AC (AG) CC (AGCT) CACT-3 ' (AG), with reverse transcription synthetic cDNA first chain is template, carry out the degenerate pcr amplification of Rtura5 gene, 10 * PCR damping fluid, 5.0 μ l, dNTPs (10mM) 1.0 μ l, ura5-sense primer (50mmol/l) 1.0 μ l, ura5-anti primer (50mmol/l) 1.0ul, rTaq enzyme (Dalian TakaRa) 0.5 μ l, the synthetic cDNA first chain template 1.0 μ l, ddH ₂O 40.5 μ l, in 94 ℃ of insulation 3min, then in 94 ℃ of 30s, 57 ℃ of 45s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.Amplified production carries out 1% (mass/volume concentration) agarose gel electrophoresis, observes the band (Fig. 1) about 0.8kb, utilizes DNA to reclaim test kit (available from the green skies), according to supplier's proposed steps purified pcr product.The method that the PCR product provides with reference to TaKaRa company is cloned into pMD18-T carrier (available from TaKaRa), be transformed into E.coli DH5 α competent cell, wherein competent cell is by Calcium Chloride Method (the molecular cloning experiment guide third edition, Sa nurse Brooker work, Huang Peitang etc. translate, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to TaKaRa company order-checking, and sequence results infers that the aminoacid sequence that analyzes through Blastp, turns out to be the orotate phosphoribosyl transferase sequence, shown in SEQ ID NO:4 sequence.Orotate phosphoribosyl transferase cDNA sequence is shown in SEQ ID NO:3 sequence.

The amplification of embodiment 3:Rtura5 gene genomic dna sequence

1.R.toruloides ATCC 10788 is (available from the biological product of USS collecting center, ATCC) extracting genome DNA adopts granulated glass sphere broken wall method (the fine works molecular biology experiment guide third edition the 13rd chapter, work such as Ao Sibai, Yan Ziying etc. translate, Science Press publishes).The genomic dna for preparing utilizes Nanodrop 1000 to measure, and records OD ₂₆₀/ OD ₂₈₀=1.85, show that the genomic dna quality is fine.Concentration is 120ng/ μ l, totally 500 μ l, and genome DNA sample is frozen in-20 ℃, and is standby.

2. according to the orotate phosphoribosyl transferase cDNA sequence that obtains among the embodiment 2, design 1 pair of gene-specific primer, ura5-ORF-p1:5 '-ATGAGCGCCACGTCCTACGC-3 ' and ura5-ORF-p2:5 '-CTAGTTAACGCCCCACTTTGCG-3 ', genomic dna with the red winter spore yeast ATCC 10788 of circle is a template, carry out pcr amplification according to ordinary method, obtain the PCR product (figure slightly) of about 0.8kb.Pcr amplification product reclaims, is cloned into the pMD18-T carrier according to the operation steps of embodiment 2, and checks order, and obtains the dna sequence dna shown in sequence table SEQ ID NO:5.Through with embodiment 2 in the orotate phosphoribosyl transferase cDNA sequence alignment that obtains, confirm that this gene fragment is its orotate phosphoribosyl transferase genomic dna sequence, intronless.

Embodiment 4: chromosome walking obtains Rtura5 gene 5 ' flank sequence (promotor)

Present embodiment utilizes Genome Walking Kit (available from TaKaRa) to finish.

According to the ura5 genomic dna sequence that obtains among the embodiment 3, design 3 Specific Primer (gene-specific primer), ura5-SP1:5 '-AGCGACGAGGGGGATGCCCTTGT-3 ', ura5-SP2:5 '-GTTGAAGAAGTAGGGCGAGGAGC-3 ' and ura5-SP3:5 '-GAGAGCGGTCTCGATGATCGAG-3 ', as downstream primer, carry out following operation according to the test kit specification sheets.

1.1 ^StThe PCR reaction

With purified genomic dna among the embodiment 3 is template, carries out first round amplification.Reaction system 50 μ l:10 * LA PCR buffer II (Mg ²⁺Plus) 5.0 μ l, dNTPs (2.5mmol/l) 8.0 μ l, LA Taq archaeal dna polymerase (5U/ μ l, Dalian TakaRa) 1.0 μ l, AP 1Primer (100 μ mol/l, Dalian TakaRa) 1.0 μ l, ura5-SP1 (10 μ mol/l) 1.0 μ l, R.toruloides ATCC 10788 genomic dna templates (120ng/ μ l) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: carry out the high specific reaction of 5 high temperature anneal temperature earlier, carry out the low specific reaction of 1 utmost point low temperature thermal oxidation then; Carry out hot asymmetric PCR then: the high specific reaction of 2 high annealing temperatures (65 ℃) and the low specific reaction alternate cycles of 1 low temperature thermal oxidation (44 ℃), totally 15 times.Concrete parameter is as follows: 94 ℃ of 1min, 98 ℃ of 1min; 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, totally 5 circulations; 94 ℃ of 30s, 25 ℃ of 3min, 72 ℃ of 2min; 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, totally 15 circulations; 72 ℃ of 10min finish reaction.

2.2 ^NdThe nest-type PRC reaction

Reaction system 50 μ l:10 * LA PCR buffer II (Mg ²⁺Plus) 5.0 μ l, dNTPs (2.5mmol/l) 8.0 μ l, LA Taq archaeal dna polymerase (5U/ μ l, Dalian TakaRa) 1.0 μ l, AP1 Primer (100 μ mol/l, Dalian TakaRa) 1.0 μ l, 1 ^StPCR reaction product 1.0 μ l, ura5-SP2 (10 μ mol/l) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, totally 15 circulations; 72 ℃ of 10min finish reaction.

3.3 ^RdThe nest-type PRC reaction

Reaction system 50 μ l:10 * LA PCR buffer II (Mg ²⁺Plus) 5.0 μ l, dNTPs (2.5mmol/l) 8.0 μ l, LA Taq archaeal dna polymerase (5U/ μ l, Dalian TakaRa) 1.0 μ l, AP1 Primer (100 μ mol/l, Dalian TakaRa) 1.0 μ l, 2 ^NdNest-type PRC reaction product 1.0 μ l, ura5-SP3 (10 μ mol/l) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, totally 15 circulations; 72 ℃ of 10min finish reaction.

3 ^RdThe nest-type PRC reaction product is cut the purpose band behind 1% (mass/volume concentration) agarose gel electrophoresis, utilize dna fragmentation gel-purified test kit (available from the green skies) to carry out purifying.Dna fragmentation behind the purifying inserts pMD18-T carrier (available from TakaRa company) through the TA clone, transforms DH5 α competent cell; Wherein competent cell is by Calcium Chloride Method (Huang Peitang etc. translate for the molecular cloning experiment guide third edition, Sa nurse Brooker work, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:1, turns out to be the pRtura5 gene order of expection.

Sequence number: 1 (SEQ ID NO:1)

Sequence length: 1000bp

Sequence type: DNA

Source: circle red winter spore yeast (Rhodosporidium toruloides)

1 TGCCGCCTAT?CTGTTAAAAC?TCAATGGATA?CAATGCAAAG?TCGGCTCGCG?CCCTCTTCTC

61 CCGTTCCCCC?GTACCTAGAC?ATGCGTCGTG?AGTGCTTCCC?TCCGTCATGC?AACCGTCGCA

121?AGCTCCTCGC?TCCGTCGTCG?CTGCTGAAGC?TGCTCCTGCG?CAATCCGCTG?CGCGGCGGGT

181?ACCTCGCCTC?GCTCCCTTTC?CGCCGCCCGA?ATCGCCTGTG?TGAGCCTGTT?CCTCCGAGAC

241?ACGACCATCG?TCTTGGGATT?GGCTTGTGGT?ACGGGCAGAG?GGCTGGTCTC?TGCAATTGAG

301?CAAGCGAGCC?GAGGCAGCGA?TGGTCAGTTC?CGGTCGCAGA?CCGAGGCTCC?AGGGAAGGGA

361?CGGCGGGACG?CACTGATGAC?GCCGGCCTGA?ATGAGCAGCT?TCTCAAAGCG?CTTTGTCGGC

421?AGCGCACCCT?GGCTGAGCCA?GTACCGGACC?CGCTCCAGGT?TCCACTCGAC?CTTCTTCTGC

481?CCGACCTTGG?CGACGCCCGT?GTTGGGCGTG?TGCTGCCGTG?GTCCCCACTC?CTTCGGGTCC

541?CTGACCTGAC?CGTTTGGCGA?GACCGACGGC?GCTGGCACGA?CGACCGGTCG?CGGGTTGTAC

601?GAACCGAGGA?GCTCGAGGGG?TTTGGCGGTC?TGGCGCTGCG?GCGCGCGGAT?GGCGACGAGC

661?GAGTAGACGG?GGTTGTTGCG?CGTGCAGTGG?TGGCGCGCGA?GGCGCAATCG?GACCGACATC

721?TCGTGCGCTG?GCGGAGAGCG?AGTGTGCGAG?GACGAGGAAA?AGGAGCGGGA?GGAGGAGGGT

781?TGGGAGCGAA?CCACCTCGTG?CAGCTGGACT?GGACGCGCTA?GACGACTGCC?AGACTCGCTA

841?TACTGCTCTT?TACATCCGCT?AGAATCCTGC?ACCCGCTCGA?ACCAGCTCCT?CCCACCGTCA

901?GTTCGCCTCC?CCCTCTCATC?CTCATCTCTC?AAAGGACTTC?CTCGCTCGCA?ACTCGCTGGA

961?GGGAGCTGCA?AGACTGACAC?GAGGTCGAAA?ACACCGTCAG

Embodiment 5: chromosome walking obtains Rtura5 gene 3 ' flank sequence (terminator)

Present embodiment also is to utilize Genome Walking Kit (available from TaKaRa) to finish.

According to the ura5DNA sequence that obtains among the embodiment 3, design 3 Specific Primer (gene-specific primer), be respectively ura5-SP11:5 '-CACGACGAGGACGTGATATCGGCT-3 ', ura5-SP22:5 '-GAGGGCGGTCAGACGGCGGGTATT-3 ' and ura5-SP33:5 '-CGTCGTCAAGATGCGCGACATCGTT-3 ', as upstream primer, carry out the operation of 3 ' flank chromosome walking according to the test kit specification sheets, remove Specific Primer respectively by ura5-SP1, ura5-SP2, ura5-SP3 is replaced by ura5-SP11 successively, ura5-SP22, outside the ura5-SP33, other is with embodiment 4.

3 ^RdThe nest-type PRC reaction product utilizes dna fragmentation gel-purified test kit (available from the green skies) to carry out purifying, inserts pMD18-T carrier (available from TakaRa company) through the TA clone, transforms DH5 α competent cell; Wherein competent cell is by Calcium Chloride Method (Huang Peitang etc. translate for the molecular cloning experiment guide third edition, Sa nurse Brooker work, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:2, turns out to be the Rtura5t gene order of expection.

Embodiment 6:Rtura5 promotor-open reading frame-terminator full-length gene obtains

Promotor and terminator sequence according to obtaining among embodiment 4 and the embodiment 5 redesign the amplification that a pair of primer carries out Rtura5 " promotor-open reading frame-terminator " full-length gene.pRtura5t-p1：5’-TGCCGCCTATCTGTTAAAACTCAATG-3’，pRtura5t-p2：5’-GCCATTCAATCTGTTCAGCCACCC-3’。R.toruloides genomic dna with preparation among the embodiment 3 is that template is carried out pcr amplification.PCR system (50 μ l): 10 * Speed buffer, 5.0 μ l, dNTPs (10mmol/l) 1.0 μ l, upstream primer Rtura5-p1 (10 μ mol/l) 2.0 μ l, downstream primer Rtura5-p2 (10 μ mol/l) 2.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (amplification rate is fast, and 1kb/10s is available from Dalian TaKaRa company) 0.5 μ l, genomic dna template (120ng/ μ l) 2 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 1.0min, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Fragment is inserted pMD18-T carrier (available from TakaRa company) through the TA clone, transforms DH5 α competent cell; Wherein competent cell is by Calcium Chloride Method (Huang Peitang etc. translate for the molecular cloning experiment guide third edition, Sa nurse Brooker work, and Science Press publishes) preparation.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction.The recombinant plasmid sample is delivered to the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:6, turns out to be the pRtura5t full length sequence of expection, this recombinant vectors called after T-pRtura5t.

Embodiment 7: the structure of the red winter spore yeast specificity egfp expression box ura5gfp of circle

The structure utilization of the red winter spore yeast specificity egfp expression box of ura5gfp circle be RF clone (Van den Ent, F., Lowe, J., 2006.RF cloning:A restriction-free method forinserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74; Yang F, Zhang S, Tang W, Zhao Z, 2008.Identification of theorotidine-5 '-monophosphate decarboxylase gene of the oleaginous yeastRhodosporidium toruloides.Yeast 25 (9): method 623-630).

Amplification of pRtura5t full length sequence and clone see embodiment 6.

1. the acquisition of green fluorescent protein encoding gene GFPuv

(available from BD Biosciences) is template with the pGFPuv plasmid, utilizes a pair of primer gfp-p1:5 '-ATGAGTAAAGGAGAAGAACT-3 ' and gfp-p2:5 '-TCATTTGTAGAGCTCATCCAT-3 ', carries out pcr amplification.System (50 μ l): 5 * Primebuffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer gfp-p1 (10 μ mol/l) 2.0 μ l, downstream primer gfp-p2 (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, pGFPuv plasmid (120ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.The PCR reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, utilize the taq archaeal dna polymerase to carry out dna fragmentation 3 ' end and add A, system (50 μ l): 10 * PCRbuffer, 5.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, GFPuv dna fragmentation 30 μ l behind the purifying, ddH ₂O adds to 50 μ l.Reaction conditions: 72 ℃ of 30min, 4 ℃ are finished reaction.The GFPuv dna fragmentation that 3 ' end adds behind the A utilizes dna fragmentation glue to reclaim the purification kit purifying, is cloned into the pMD18-T carrier, send the order-checking of TaKaRa company, obtains the dna sequence dna shown in SEQ ID NO:7, turns out to be the GFPuv gene order of expection.

2. reference literature method (Van den Ent, F., Lowe, J., 2006.RF cloning:Arestriction-free method for inserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74), design RF clone primer: ura5-gfp-p1:5 '-CACGAGGTCGAAAACACCGTCAG atgagtaaaggagaagaact-3 ' and ura5-gfp-p2:5 '-GGAACTGCCCAGACGCACTTGTAT ctatttgtagagctcatccat-3 ' (wherein original ura5ORF flank sequence complementation in capitalization partial sequence and the pRtura5t cloning vector, lowercase partial sequence and green fluorescent protein GFPuv ORF complementation).

3.RF I reaction system and flow process: the GFPuvTA cloning vector with structure in the present embodiment action-item 1 is a template, utilizes ura5gfp-p1 and ura5gfp-p2 to be primer, carries out the RF first round and increases.System (50 μ l): 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer (10 μ mol/l) 2.0 μ l, downstream primer (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, T-GFPuv plasmid (100ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.RF I reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, and-20 ℃ of preservations are standby.

4.RF II reaction: 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, T-pRtura5t plasmid (100ng/ μ l) the 1.0 μ l that make up among the embodiment 6, RFI reaction product in the present embodiment abovementioned steps (100ng/ μ l) 5.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 68 ℃ of 12min, 95 ℃ of 30s afterwards, 65 ℃ of 45s (1 ℃/cyc), and 68 ℃ of 12min, 15 circulations, next carry out again taking turns: 95 ℃ of 30s, 55 ℃ of 45s, 68 ℃ of 12min, 20 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

5.DpnI digestion and electric shock transform: get 8 μ l RF II reaction product add behind 1 μ l DpnI (available from New England Biolabs) and the 1 μ l DpnI buffer mixing 37 ℃ act on 120min and remove former T-pRtura5t plasmid after, get 2 μ l electric shock respectively and transform DH5 α competent cell, competent cell is by standard method preparation (the molecular cloning experiment guide third edition, Sa nurse Brooker work, Huang Peitang etc. translate, Science Press publishes), electric shock transforms parameter: 2200-2500V, 400 Ω, 25 μ F, 0 ℃.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction, and utilize RF I reaction the primer ura5gfp-p1 and ura5gfp-p2 to carry out bacterium colony PCR and identify, identify that the male recombinant vectors send TaKaRa to check order, obtain the ura5gfp expression cassette that 5 ' end and 3 ' end are respectively ura5 promotor and ura5 terminator, simultaneously, this recombinant vectors called after pura5gfp.GFPuv ORF sequence is shown in SEQ ID NO:7; Complete ura5gfp expression cassette is shown in SEQ ID NO:8.

Embodiment 8: utilize the ura5gfp expression cassette to carry out the Rtura5 gene knockout egfp expression of holding concurrently

1.Rtura5gfp knock out the preparation of box

The pura5gfp carrier that makes up with embodiment 7 is a template, is primer with oligonucleotide sequence pRtura5t-p1 among the embodiment 6 and pRtura5t-p2, carries out a large amount of preparations that Rtura5gfp knocks out box.PCR system (500 μ l): 10 * Speed buffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (Dalian TaKaRa company) 5.0 μ l, genomic dna template (120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Dna fragmentation concentration behind the purifying is at 380ng/ μ l, totally 50 μ l, and-20 ℃ of preservations are standby.

2. the red winter spore yeast ATCC 10788 competent cells preparation of monoploid circle

The preparation of R.toruloides np11 competent cell: R.toruloides ATCC 10788 (available from the biological product ATCC of collecting center of USS) bacterial strain is chosen colony inoculation 10ml YEPD substratum (glucose 20.0g/l, yeast extract 10.0g/l, peptone 20.0g/l, pH 6.0), 30 ℃, 200rpm cultivates 20h; 1: 50 ratio of culture fresh YEPD substratum of transferring, 100ml (500ml Erlenmeyer flask, liquid amount 100ml), 30 ℃, 200rpm cultivates 6-9h, and the OD value reaches 0.6-1.2; Culture ice bath 10-30min, 4 ℃, the centrifugal 5min of 4000rpm abandons supernatant; 0 ℃ of aseptic Milli-Q washes 1 time; 0 ℃ of 1mol/l sorbyl alcohol washs 2 times; Ice bath, standby.

3. justifying the electric shock conversion of red winter spore yeast ATCC 10788 and the PCR of transformant identifies

The electric shock that Rtura5gfp knocks out box transforms: get 100 μ l R.toruloides ATCC, 10788 competent cells, add Rtura5gfp and knock out box 5 μ l (2 μ g altogether), ice bath 5min behind the mixing, move in the electric shock cup that is chilled to 0 ℃ in advance, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add 1ml YEPD after the electric shock immediately, 30 ℃ of incubation 1-2h; Coating YEPD flat board, 10 μ l/ flat boards are cultivated 28-36h for 30 ℃; Choose mono-clonal one by one and utilize fluorescent microscope to carry out microscopy, the fluorescence photograph of positive recombinant ATCC 10788 Δ ura5::gfp as shown in Figure 5.

The red winter spore yeast ATCC 10788 Δ ura5::gfp YEPD of 3 strains reorganization circle cultivate the culture of 24h, utilize physiological saline (0.9%NaCl damping fluid) to carry out doubling dilution, select its 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6Four extent of dilution pipette 10 μ l bacterium liquid respectively in the SC solid medium that contains 5 '-FOA (5 '-fluororotic acid is available from Shanghai Jinhe Biotechnology Co., Ltd) (0.2%5 '-FOA, glucose 70g/L, (NH ₄) ₂SO ₄0.1g/L, yeast powder 0.75g/L, KH ₂PO ₄0.4g/L, MgSO ₄7H ₂O1.5g/L, pH 6.0) flat board, treat that liquid-absorbent is complete, be inverted in 30 ℃ and cultivated 2 days, find that the red winter spore yeast ATCC 10788 Δ ura5::gfp of reorganization circle possess 5 '-FOA resistance, the red winter spore yeast ATCC 10788 then aseptic length of being born of control strain circle.

Above presentation of results, Rtura5gfp knock out box (expression cassette) when starting the conformability of green fluorescent protein in the red winter spore yeast of circle and expressing, and can deactivation integrate the orotidine-5 activity of position ura5 genes encoding.Such design helps the genetic manipulation in later stage.

Embodiment 9: the structure of the red winter spore yeast specificity bleomycin resistance expression box ura5ble of circle

The structure of the red winter spore yeast specificity bleomycin expression cassette of ura5ble circle is the method for utilizing the RF clone equally.Amplification of pRtura5t full length sequence and clone see embodiment 6.

1. reference literature method (Van den Ent, F., Lowe, J., 2006.RF cloning:Arestriction-free method for inserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74), design RF clone primer: ura5-ble-p1:5 '-CACGAGGTCGAAAACACCGTCAG atggccaagttgaccagtgccgtt-3 ' and ura5-ble-p2:5 '-GGAACTGCCCAGACGCACTT GTATctagtcctgctcctcggccacg-3 ' (wherein original ura5ORF flank sequence complementation in capitalization partial sequence and the pRtura5t cloning vector, lowercase part and bleomycin resistant gene ble ORF complementation).

2.RF I reaction system and flow process: with pPICZ α A plasmid (available from Invitrogen company) is template, utilizes ura5ble-p1 and ura5ble-p2 to be primer, carries out the RF first round and increases.System (50 μ l): 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer (10 μ mol/l) 2.0 μ l, downstream primer (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, pPICZ α A plasmid (100ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.RF I reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, and-20 ℃ of preservations are standby.

3.RF II reaction: 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, T-pRtura5t plasmid (100ng/ μ l) the 1.0 μ l that make up among the embodiment 6, RFI reaction product in the present embodiment abovementioned steps (100ng/ μ l) 5.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 68 ℃ of 12min, 95 ℃ of 30s afterwards, 65 ℃ of 45s (1 ℃/cyc), and 68 ℃ of 12min, 15 circulations, next carry out again taking turns: 95 ℃ of 30s, 55 ℃ of 45s, 68 ℃ of 12min, 20 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

4.DpnI digestion and electric shock transform: get 8 μ l RF II reaction product add behind 1 μ l DpnI (available from New England Biolabs) and the 1 μ l DpnI buffer mixing 37 ℃ act on 120min and remove former T-pRtura5t plasmid after, get 2 μ l electric shock respectively and transform DH5 α competent cell, competent cell is by standard method preparation (the molecular cloning experiment guide third edition, Sa nurse Brooker work, Huang Peitang etc. translate, Science Press publishes), electric shock transforms parameter: 2200-2500V, 400 Ω, 25 μ F, 0 ℃.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction, and utilize RF I reaction the primer ura5ble-p1 and ura5ble-p2 to carry out bacterium colony PCR and identify, identify that the male recombinant vectors send TaKaRa to check order, obtain the ura5ble expression cassette that 5 ' end and 3 ' end are respectively ura5 promotor and ura5 terminator, simultaneously, this recombinant vectors called after pura5ble.Ble ORF sequence is shown in SEQ ID NO:9; Complete ura5ble expression cassette is shown in SEQ ID NO:10.

Embodiment 10: utilize the ura5ble expression cassette to carry out the Rtura5 gene knockout bleomycin resistance expression that holds concurrently

1.Rtura5ble knock out the preparation of box

The pura5ble carrier that makes up with embodiment 9 is a template, is primer with oligonucleotide sequence pRtura5t-p1 among the embodiment 6 and pRtura5t-p2, carries out a large amount of preparations that Rtura5ble knocks out box.PCR system (500 μ l): 10 * Speed buffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (Dalian TaKaRa company) 5.0 μ l, genomic dna template (120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Dna fragmentation concentration behind the purifying is at 300ng/ μ l, totally 50 μ l, and-20 ℃ of preservations are standby.

2. the red winter spore yeast ATCC 10788 competent cells preparation of monoploid circle

The preparation of R.toruloides np11 competent cell is with the action-item among the embodiment 83.

3. justifying the electric shock conversion of red winter spore yeast ATCC 10788 and the PCR of transformant identifies

The electric shock that Rtura5ble knocks out box transforms: get 100 μ l R.toruloides ATCC, 10788 competent cells, add Rtura5ble and knock out box 10 μ l (3 μ g altogether), move in the electric shock cup that is chilled to 0 ℃ in advance behind the mixing, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add 1ml YEPD after the electric shock immediately, 30 ℃ of incubation 1-2h; Coating contains the YEPD flat board of 20 μ g/ml Zeocin (available from Invitrogen company), and 200 μ l/ flat boards are cultivated 2-10d, had 100 transformants to occur successively approximately for 30 ℃.

3 Zeocin resistances of picking transformant is cultivated 24h in the YEPD substratum, culture utilizes physiological saline (0.9%NaCl damping fluid) to carry out doubling dilution, select its 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6Four extent of dilution pipette 10 μ l bacterium liquid spottings respectively in the SC solid medium that contains 5 '-FOA (available from Shanghai Jinhe Biotechnology Co., Ltd) (0.2%5 '-FOA, glucose 70g/L, (NH ₄) ₂SO ₄0.1g/L, yeast powder 0.75g/L, KH ₂PO ₄0.4g/L, MgSO ₄7H ₂O 1.5g/L, pH 6.0) flat board, treat that liquid-absorbent fully after, be inverted in 30 ℃ and cultivated 3 days, find that the red winter spore yeast ATCC 10788 Δ ura5::ble of reorganization circle possess 5 '-FOA resistance, the then aseptic length of being born of the control strain red winter spore yeast ATCC10788 of circle.

Above presentation of results, Rtura5ble knock out box (expression cassette) when starting the bleomycin resistant gene conformability is expressed in the red winter spore yeast of circle, can deactivation integrate the orotidine-5 activity of position ura5 genes encoding.Such design helps the genetic manipulation in later stage.

Embodiment 11: the structure of the red winter spore yeast specificity Geneticin resistance expression box ura5kan of circle

The structure of the red winter spore yeast specificity Geneticin resistance expression box of ura5kan circle is the method for utilizing the RF clone equally.Amplification of pRtura5t full length sequence and clone see embodiment 6.

1. reference literature method (Van den Ent, F., Lowe, J., 2006.RF cloning:Arestriction-free method for inserting target genes into plasmids.J.Biochem.Biophys.Methods 67:67-74), design RF clone primer: ura5-kan-p1:5 '-CACGAGGTCGAAAACACCGTCAG atgggtaaggaaaagactcacgt-3 ' and ura5-kan-p2:5 '-GGAACTGCCCAGACGCACTT GTATttagaaaaactcatcgagcatc-3 ' (wherein original ura5ORF flank sequence complementation in capitalization partial sequence and the pRtura5t cloning vector, lowercase partial sequence and Geneticin resistant gene kanmx4ORF complementation).

2.RF I reaction system and flow process: (available from EUROSCARF) is template with the pFA6-kanmx4 plasmid, utilizes ura5kan-p1 and ura5kan-p2 to be primer, carries out the RF first round and increases.System (50 μ l): 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, upstream primer (10 μ mol/l) 2.0 μ l, downstream primer (10 μ mol/l) 2.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, pFA6-kanmx4 plasmid (110ng/ μ l) 1 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 98 ℃ of 8s, 49 ℃ of 15s, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.RF I reaction product utilizes dna fragmentation glue to reclaim the purification kit purifying, and-20 ℃ of preservations are standby.

3.RF II reaction: 5 * Prime buffer, 10.0 μ l, dNTPs (2.5mmol/l) 4.0 μ l, T-pRtura5t plasmid (100ng/ μ l) the 1.0 μ l that make up among the embodiment 6, RFI reaction product in the present embodiment abovementioned steps (100ng/ μ l) 5.0 μ l, PrimeSTAR ^TMHS archaeal dna polymerase (Dalian TakaRa) 1.0 μ l, ddH ₂O adds to 50 μ l.Reaction conditions: 95 ℃ of 3min, 68 ℃ of 12min, 95 ℃ of 30s afterwards, 65 ℃ of 45s (1 ℃/cyc), and 68 ℃ of 12min, 15 circulations, next carry out again taking turns: 95 ℃ of 30s, 55 ℃ of 45s, 68 ℃ of 12min, 20 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

4.DpnI digestion and electric shock transform: get 8 μ l RF II reaction product add behind 1 μ l DpnI (available from New England Biolabs) and the 1 μ l DpnI buffer mixing 37 ℃ act on 120min and remove former T-pRtura5t plasmid after, get 2 μ l electric shock respectively and transform DH5 α competent cell, competent cell is by standard method preparation (the molecular cloning experiment guide third edition, Sa nurse Brooker work, Huang Peitang etc. translate, Science Press publishes), electric shock transforms parameter: 2200-2500V, 400 Ω, 25 μ F, 0 ℃.Select Amp resistance transformant and increase bacterium cultivation, plasmid extraction, and utilize RF I reaction the primer ura5kan-p1 and ura5kan-p2 to carry out bacterium colony PCR and identify, identify that the male recombinant vectors send TaKaRa to check order, obtain the ura5kan expression cassette that 5 ' end and 3 ' end are respectively ura5 promotor and ura5 terminator, simultaneously, this recombinant vectors called after pura5kan.Kanmx ORF sequence is shown in SEQID NO:11; Complete ura5kan expression cassette is shown in SEQ ID NO:12.

Embodiment 12: utilize the ura5kan expression cassette to carry out the Rtura5 gene knockout Geneticin resistance expression that holds concurrently

1.Rtura5kan knock out the preparation of box

The pura5kan carrier that makes up with embodiment 11 is a template, is primer with oligonucleotide sequence pRtura5t-p1 among the embodiment 6 and pRtura5t-p2, carries out a large amount of preparations that Rtura5kan knocks out box.PCR system (500 μ l): 10 * Speed buffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase (Dalian TaKaRa company) 5.0 μ l, genomic dna template (120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Dna fragmentation concentration behind the purifying is at 300ng/ μ l, totally 40 μ l, and-20 ℃ of preservations are standby.

2. the red winter spore yeast ATCC 10788 competent cells preparation of monoploid circle

The preparation of R.toruloides np11 competent cell is with the action-item among the embodiment 83.

3. justifying the electric shock conversion of red winter spore yeast ATCC 10788 and the PCR of transformant identifies

The electric shock that Rtura5kan knocks out box transforms: get 100 μ l R.toruloides ATCC, 10788 competent cells, add Rtura5kan and knock out box 10 μ l (3.2 μ g altogether), ice bath 5min behind the mixing, move in the electric shock cup that is chilled to 0 ℃ in advance, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add 1ml YEPD after the electric shock immediately, 30 ℃ of incubation 1-2h; Coating contains the YEPD flat board of 50 μ g/mlG418 (available from Beijing boat ancient cooking vessel state), and 200 μ l/ flat boards are cultivated 2-10d, had 60 transformants to occur successively approximately for 30 ℃.

3 G418 resistances of picking transformant is cultivated 24h in the YEPD substratum, culture utilizes physiological saline (0.9%NaCl damping fluid) to carry out doubling dilution, select its 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6Four extent of dilution pipette 10 μ l bacterium liquid spottings respectively in the SC solid medium that contains 5 '-FOA (available from Shanghai Jinhe Biotechnology Co., Ltd) (0.2%5 '-FOA, glucose 70g/L, (NH ₄) ₂SO ₄0.1g/L, yeast powder 0.75g/L, KH ₂PO ₄0.4g/L, MgSO ₄7H ₂O 1.5g/L, pH 6.0) flat board, treat that liquid-absorbent fully after, be inverted in 30 ℃ and cultivated 3 days, find that the red winter spore yeast ATCC 10788 Δ ura5::kan of reorganization circle possess 5 '-FOA resistance, the then aseptic length of being born of the control strain red winter spore yeast ATCC10788 of circle.

Above presentation of results, Rtura5kan knock out box (expression cassette) when starting the Geneticin resistant gene conformability is expressed in the red winter spore yeast of circle, can deactivation integrate the orotidine-5 activity of the ura5 genes encoding of position.Such design helps the genetic manipulation in later stage.

(not of the same race) determination of activity in the genus of embodiment 13:pRtura5 promotor and Rtura5t terminator

Also be of the functional verifications of expression cassettes such as ura5gfp, ura5ble, ura5kan at R.babjevae.

Utilize the 26SrDNA gene as integration site at this, make each integration expression box 5 ' end carry the 26SrDNA dna homolog reorganization arm of the 500bp that has an appointment by merging PCR method, carry out ura5gfp expression cassette, ura5ble expression cassette, the ura5kan expression cassette conformability in R.babjevae respectively and express.

1. the R.babjevae 26SrDNA sequence of announcing according to NCBI (NO.:EF595746), design a pair of primer: Rb26S-Rtura5-p1:5 '-AGCGGCGAGCGAAGCGGTAAG-3 ' and Rb26S-Rtura5-p2:5 '-gagttttaacagataggcggcaACGCTGCGTTCCTCAGTCCCC-3 ' (wherein capitalization partial sequence and R.babjevae 26SrDNA sequence 3 ' are held homology, and the lowercase partial sequence is complementary with the red winter spore yeast pura5 promotor 5 ' end of circle).

2.Rb26S-Rtura5gfp, the structure of Rb26S-Rtura5ble, Rb26S-Rtura5kan

Utilize Pura5gfp, Pura5ble and Pura5kan carrier among embodiment 5, embodiment 7 and the embodiment 9 to be template respectively, carry out the structure of 3 amalgamation and expression boxes such as Rb26S-Rtura5gfp, Rb26S-Rtura5ble, Rb26S-Rtura5kan respectively.PCR system (each 500 μ l): 10 * Speedbuffer, 50.0 μ l, dNTPs (10mmol/l) 10.0 μ l, upstream primer (10 μ mol/l) 20.0 μ l, downstream primer (10 μ mol/l) 20.0 μ l, SpeedSTAR ^TMHS archaeal dna polymerase 5.0 μ l, dna profiling plasmid Pura5gfp or Pura5ble or Pura5kan (all 120ng/ μ l) 15.0 μ l, ddH ₂O adds to 500 μ l.Reaction conditions: 98 ℃ of 1min, 98 ℃ of 10s, 65 ℃ of 60s, 35 circulations, 72 ℃ of 10min, 4 ℃ are finished reaction.

The PCR product utilizes PCR fragment purification test kit (available from the green skies) to carry out purifying after 1% (mass/volume concentration) agarose gel electrophoresis analysis.Rb26S-Rtura5gfp behind the purifying, Rb26S-Rtura5ble and Rb26S-Rtura5kan expression cassette concentration are respectively 310ng/ μ l, 300ng/ μ l and 270ng/ μ l, equal 45 μ l, and-20 ℃ of preservations are standby.

3.R.babjevae ATCC90942 competent cell preparation

R.babjevae ATCC90942 is available from available from the biological product of USS collecting center.At first, choose colony inoculation 10ml YEPD substratum, 28 ℃, 200rpm cultivates 24h; 1: 50 ratio of culture fresh YEPD substratum of transferring, 100ml (500ml Erlenmeyer flask, liquid amount 100ml), 28 ℃, 200rpm cultivates 7-10h, and the OD value reaches 0.6-1.2; Culture ice bath 10-30min, 4 ℃, the centrifugal 5min of 4000rpm abandons supernatant; 0 ℃ of aseptic Milli-Q washes 1 time; 0 ℃ of 1mol/l sorbyl alcohol washs 2 times; Ice bath, standby.

4.R.babjevae the electric shock of ATCC90942 transforms and expression of results is observed

Get 3 part of 100 μ l R.babjevae ATCC90942 competent cell, add each 10 μ l of Rb26S-Rtura5gfp, Rb26S-Rtura5ble and Rb26S-Rtura5kan expression cassette respectively, move in the electric shock cup that is chilled to 0 ℃ in advance behind the mixing, voltage 0.8-2.0 kilovolt, resistance 200 Ω, electric capacity 25 μ F, time 4-10ms; Add 1ml YEPD after the electric shock immediately, 30 ℃ of incubation 1-2h; Rb26S-Rtura5gfp conversion fluid coating YEPD flat board, 10 μ l/ flat boards; The coating of Rb26S-Rtura5ble conversion fluid contains the YEPD flat board of 20 μ g/mlZeocin (available from Invitrogen company), 200 μ l/ flat boards; The coating of Rb26S-Rtura5kan conversion fluid contains the YEPD flat board of 50 μ g/ml G418 (available from Beijing boat ancient cooking vessel state), 200 μ l/ flat boards; Cultivate 2-10d for equal 28 ℃.

R.babjevae ATCC90942/Rb26S-Rtura5gfp transformant is chosen mono-clonal one by one and is utilized fluorescent microscope to carry out microscopy, and the positive recombinant of a luciferase expression can be arranged in per 40 transformants, and the fluorescence photograph slightly; The coating of Rb26S-Rtura5ble conversion fluid contains the YEPD flat board of 20 μ g/ml Zeocin, can be observed resistance recon not of uniform size during 28 ℃ of cultivation 5d, and average 100/flat board transforms 200 recons of recombination efficiency/μ g expression cassette DNA; The coating of Rb26S-Rtura5kan conversion fluid contains the YEPD flat board of 50 μ g/ml G418, can be observed resistance recon not of uniform size during 28 ℃ of cultivation 6d, and average 100/flat board transforms 200 recons of recombination efficiency/μ g expression cassette DNA.

Each expression cassette is at the apparent conversion recombination efficiency of R.babjevae ATCC90942, than the height in R.toruloides ATCC 10788, may be because what carry out at R.babjevae ATCC90942 is the unit point homologous recombination, and what carry out at R.toruloides ATCC10788 be the reason of dibit point homologous recombination.

Above embodiment proves that the ura5 promotor can start green fluorescence protein gene, bleomycin resistant gene and the Geneticin resistant gene conformability in R.toruloides ATCC 10788 and express; Ura5gfp expression cassette, ura5ble expression cassette, ura5kan expression cassette are that the linear DNA of fundamental construction knocks out box, can knock out the ura5 target gene on the red winter spore yeast genes group of circle.Use the dna molecular with promoter activity of the present invention and have the active DNA of transcription terminator, can realize foreign gene or native gene in the expression of circle in the red winter spore yeast, the invention provides and be used for genetic engineering circle red winter spore zymic promotor, terminator and a series of DNA construct.For the red winter spore yeast of circle has been opened a breeding new way, and therefore can provide the spore yeast of the red winter of novel circle with industrial use.And method and platform are provided for other saccharomycetic genetic manipulation of Rhodosporidium.

Embodiment 14: the structural characterization of the red winter spore yeast orotate phosphoribosyl transferase OPRTase of circle

The reference molecule cloning experimentation guide third edition (Sa nurse Brooker work, Huang Peitang etc. translate, Science Press publishes) obtain the red winter spore yeast OPRTase of circle through size-exclusion, ion-exchange purification.The OPRTase crystal at room temperature obtains by sessile drop method.Adopt the sparse matrix sampling method, grope to obtain to satisfy high resolution structures and resolve the crystalline parameter that requires.Final crystallization condition is as follows: RtFBA protein solution (15mg/ml) and isopyknic mother liquor (0.1M Tris-HCl (pH 7.4), 0.08M magnesium acetate, 26% (w/v) polyethylene glycol 6000) mix, and 2-3 crystal occurs in week.Determine the position phase with multi-wavelength anomalous scattering method (MAD), the MAD data are collected on ADSC Quantum-4R ccd detector, and all data are unified with the DPS software package, carry out coordinate correction and processing with the CCP4 software package.Model makes up on Silicon Graphics OCTANE and proofreaies and correct with XtalView 4.0 softwares, refines with the REFMAC program.The red winter spore yeast OPRTase crystal of circle belongs to spacer P4 ₃2 ₁2, lattice parameter is

Obtain 3 d structure model according to diffraction data and see Figure 12.

The invention has the beneficial effects as follows:

For the yeast of justifying red winter spore yeast or Rhodosporidium provides promotor, terminator, selected marker's expression cassette and genetic transforming method, with the promotion spore yeast of red winter of circle from now on or the saccharomycetic strain improvement research of Rhodosporidium effectively, accelerate the red winter spore yeast metabolism engineering research of circle.

SEQ?ID?NO：1

TGCCGCCTAT?CTGTTAAAAC?TCAATGGATA?CAATGCAAAG?TCGGCTCGCG?CCCTCTTCTC 60

CCGTTCCCCC?GTACCTAGAC?ATGCGTCGTG?AGTGCTTCCC?TCCGTCATGC?AACCGTCGCA 120

AGCTCCTCGC?TCCGTCGTCG?CTGCTGAAGC?TGCTCCTGCG?CAATCCGCTG?CGCGGCGGGT 180

ACCTCGCCTC?GCTCCCTTTC?CGCCGCCCGA?ATCGCCTGTG?TGAGCCTGTT?CCTCCGAGAC 240

ACGACCATCG?TCTTGGGATT?GGCTTGTGGT?ACGGGCAGAG?GGCTGGTCTC?TGCAATTGAG 300

CAAGCGAGCC?GAGGCAGCGA?TGGTCAGTTC?CGGTCGCAGA?CCGAGGCTCC?AGGGAAGGGA 360

CGGCGGGACG?CACTGATGAC?GCCGGCCTGA?ATGAGCAGCT?TCTCAAAGCG?CTTTGTCGGC 420

AGCGCACCCT?GGCTGAGCCA?GTACCGGACC?CGCTCCAGGT?TCCACTCGAC?CTTCTTCTGC 480

CCGACCTTGG?CGACGCCCGT?GTTGGGCGTG?TGCTGCCGTG?GTCCCCACTC?CTTCGGGTCC 540

CTGACCTGAC?CGTTTGGCGA?GACCGACGGC?GCTGGCACGA?CGACCGGTCG?CGGGTTGTAC 600

GAACCGAGGA?GCTCGAGGGG?TTTGGCGGTC?TGGCGCTGCG?GCGCGCGGAT?GGCGACGAGC 660

GAGTAGACGG?GGTTGTTGCG?CGTGCAGTGG?TGGCGCGCGA?GGCGCAATCG?GACCGACATC 720

TCGTGCGCTG?GCGGAGAGCG?AGTGTGCGAG?GACGAGGAAA?AGGAGCGGGA?GGAGGAGGGT 780

TGGGAGCGAA?CCACCTCGTG?CAGCTGGACT?GGACGCGCTA?GACGACTGCC?AGACTCGCTA 840

TACTGCTCTT?TACATCCGCT?AGAATCCTGC?ACCCGCTCGA?ACCAGCTCCT?CCCACCGTCA 900

GTTCGCCTCC?CCCTCTCATC?CTCATCTCTC?AAAGGACTTC?CTCGCTCGCA?ACTCGCTGGA 960

GGGAGCTGCA?AGACTGACAC?GAGGTCGAAA?ACACCGTCAG 1000

SEQ?ID?NO：2

ATACAAGTGC?GTCTGGGCAG?TTCCCCTCTT?TTGCGTAGCG?TACCCGCACG?ATTTACTGAC 60

GCAACTCGGG?CGCTACAGTC?CTGTCAATCT?ATCGCGATCT?TCGCAGCGAC?ACTCGCCGCT 120

TCCGATTCCC?GACTTTGCGT?CCATCTCGCT?CGCTTCGATA?AGCTTCTTTG?GCTGCTACAG 180

ACGGCTCTCA?ACTCTGCTGG?AAGATGCAGC?ATCAGCACTC?ACGGGCTCCG?CAACAGCTGC 240

TGTCACGCCT?CGAGTTCAGT?CGGGCAGCTC?TCGCCATCTC?GCAACCCGTT?ATGGCGATGG 300

TCGACCCGTC?TCGCGGCTTT?CCCAGCGCTG?CGTTTCCGCT?TCAGCTGTCG?CGGTACAAAC 360

TTCATCGCCC?CGTAGCGAGC?CGTCTCTCGC?GCAACGAGGC?AATCTCGCGC?TTCTCTCCCT 420

AACGCTTTCT?ATCGGTCCCG?GCCTCTCTCC?CGCGGACTCT?AAGAGACGTC?GACGCAGTTT 480

CGATATCGTC?AACAGCGAGC?GCAGACGAGC?ACTGCCCGAG?CAGTGAACGA?CGACGAGCTC 540

TGTACGAAGC?GATCGGATCC?GCCGCCTTCG?CTCGCGACAT?GCTCGAGCTT?TCCTGATCAC 600

CTTGTCTGAG?TTGCTCAGGC?TCTGCTAAGT?CAGTCGTGCG?CATCGGAGTT?TGCTGAGGCG 660

CGAGAAGGAT?ATTACAGCTC?GTACGAAGGA?CGGCGACTCT?GGCTCGCTGT?CCGTCACACG 720

AGACAGAGGC?CAGCCTCGAC?CTCGTCAGCG?AGTCGGTCTG?TCCATGACGG?CTAACTAATG 780

AGGGTGGCTG?AACAGATTGA?ATGGC 805

SEQ ID NO:3 (the red winter spore yeast orotate phosphoribosyl transferase cDNA sequence of circle)

ATGAGCGCCA?CGTCCTACGC?CGCCTCGATC?ATCGAGACCG?CTCTCGCGAG?CGAGCAGCCC 60

ATCCTCCGCT?TCGGCACCTT?CACCCTCAAG?TCTGGCCGCT?CCTCGCCCTA?CTTCTTCAAC 120

TTTGGCCTCT?TCAACACCGG?CTCTCTCCTC?CTCGCTCTCG?CCTCGGCCTT?CGCAGACGCC 180

ATCCTCGACG?CCTACCCCGA?GATTGGCTCC?TCCTCCGCCG?GTCCCGACAC?GCCCAAAGTC 240

CTCTTCGGAC?CCGCCTACAA?GGGCATCCCC?CTCGTCGCTG?CTATCGCATC?CGAACTCGCA 300

CGACGAGGAC?GTGATATCGG?CTACAGCTAC?AACCGCAAGG?AGAAGAAGGA?CCACGGCGAG 360

GGCGGGTCGA?TTGTCGGTGC?GCCGCTCAAG?GGACAGAAGG?TCCTCATCGT?CGACGACGTG 420

ATCACGGCCG?GTACTGCGAT?TCGCGAGGCG?CACAAGATCG?TCGAGTCGGA?GGGCGGTCAG 480

ACGGCGGGTA?TTGTCGAGGC?CCTCGACCGG?GAGGAGCGCG?GACAGGGCGA?GCTCAGTACG 540

GTCCAGGAAG?TCGAGAAGGA?GCTCGGCGTC?AAGGTCACGA?GCGTCGTCAA?GATGCGCGAC 600

ATCGTTGCGT?GGCTCAAAGA?GAAGGGCAAG?CTCGACGAGA?TGAAGGCCAT?GGAGGAGTAC 660

CGCGCAAAGT?GGGGCGTTAA?CTAG 684

SEQ?ID?NO：4

Met?Ser?Ala?Thr?Ser?Tyr?Ala?Ala?Ser?Ile?Ile?Glu?Thr?Ala?Leu

Ala?Ser?Glu?Gln?Pro?Ile?Leu?Arg?Phe?Gly?Thr?Phe?Thr?Leu?Lys

Ser?Gly?Arg?Ser?Ser?Pro?Tyr?Phe?Phe?Asn?Phe?Gly?Leu?Phe?Asn

Thr?Gly?Ser?Leu?Leu?Leu?Ala?Leu?Ala?Ser?Ala?Phe?Ala?Asp?Ala

Ile?Leu?Asp?Ala?Tyr?Pro?Glu?Ile?Gly?Ser?Ser?Ser?Ala?Gly?Pro

Asp?Thr?Pro?Lys?Val?Leu?Phe?Gly?Pro?Ala?Tyr?Lys?Gly?Ile?Pro

Leu?Val?Ala?Ala?Ile?Ala?Ser?Glu?Leu?Ala?Arg?Arg?Gly?Arg?Asp

Ile?Gly?Tyr?Ser?Tyr?Asn?Arg?Lys?Glu?Lys?Lys?Asp?His?Gly?Glu

Gly?Gly?Ser?Ile?Val?Gly?Ala?Pro?Leu?Lys?Gly?Gln?Lys?Val?Leu

Ile?Val?Asp?Asp?Val?Ile?Thr?Ala?Gly?Thr?Ala?Ile?Arg?Glu?Ala

His?Lys?Ile?Val?Glu?Ser?Glu?Gly?Gly?Gln?Thr?Ala?Gly?Ile?Val

Glu?Ala?Leu?AsP?Arg?Glu?Glu?Arg?Gly?Gln?Gly?Glu?Leu?Ser?Thr

Val?Gln?Glu?Val?Glu?Lys?Glu?Leu?Gly?Val?Lys?Val?Thr?Ser?Val

Val?Lys?Met?Arg?Asp?Ile?Val?Ala?Trp?Leu?Lys?Glu?Lys?Gly?Lys

Leu?Asp?Glu?Met?Lys?Ala?Met?Glu?Glu?Tyr?Arg?Ala?Lys?Trp?Gly

Val?Asn

SEQ ID NO:5 (the red winter spore yeast whey acid phosphoric acid ribose transferase gene group dna sequence dna of circle)

ATGAGCGCCA?CGTCCTACGC?CGCCTCGATC?ATCGAGACCG?CTCTCGCGAG?CGAGCAGCCC 60

ATCCTCCGCT?TCGGCACCTT?CACCCTCAAG?TCTGGCCGCT?CCTCGCCCTA?CTTCTTCAAC 120

TTTGGCCTCT?TCAACACCGG?CTCTCTCCTC?CTCGCTCTCG?CCTCGGCCTT?CGCAGACGCC 180

ATCCTCGACG?CCTACCCCGA?GATTGGCTCC?TCCTCCGCCG?GTCCCGACAC?GCCCAAAGTC 240

CTCTTCGGAC?CCGCCTACAA?GGGCATCCCC?CTCGTCGCTG?CTATCGCATC?CGAACTCGCA 300

CGACGAGGAC?GTGATATCGG?CTACAGCTAC?AACCGCAAGG?AGAAGAAGGA?CCACGGCGAG 360

GGCGGGTCGA?TTGTCGGTGC?GCCGCTCAAG?GGACAGAAGG?TCCTCATCGT?CGACGACGTG 420

ATCACGGCCG?GTACTGCGAT?TCGCGAGGCG?CACAAGATCG?TCGAGTCGGA?GGGCGGTCAG 480

ACGGCGGGTA?TTGTCGAGGC?CCTCGACCGG?GAGGAGCGCG?GACAGGGCGA?GCTCAGTACG 540

GTCCAGGAAG?TCGAGAAGGA?GCTCGGCGTC?AAGGTCACGA?GCGTCGTCAA?GATGCGCGAC 600

ATCGTTGCGT?GGCTCAAAGA?GAAGGGCAAG?CTCGACGAGA?TGAAGGCCAT?GGAGGAGTAC 660

CGCGCAAAGT?GGGGCGTTAA?CTAG 684

SEQ?ID?NO：6

TGCCGCCTAT?CTGTTAAAAC?TCAATGGATA?CAATGCAAAG?TCGGCTCGCG?CCCTCTTCTC 60

CCGTTCCCCC?GTACCTAGAC?ATGCGTCGTG?AGTGCTTCCC?TCCGTCATGC?AACCGTCGCA 120

AGCTCCTCGC?TCCGTCGTCG?CTGCTGAAGC?TGCTCCTGCG?CAATCCGCTG?CGCGGCGGGT 180

ACCTCGCCTC?GCTCCCTTTC?CGCCGCCCGA?ATCGCCTGTG?TGAGCCTGTT?CCTCCGAGAC 240

ACGACCATCG?TCTTGGGATT?GGCTTGTGGT?ACGGGCAGAG?GGCTGGTCTC?TGCAATTGAG 300

CAAGCGAGCC?GAGGCAGCGA?TGGTCAGTTC?CGGTCGCAGA?CCGAGGCTCC?AGGGAAGGGA 360

CGGCGGGACG?CACTGATGAC?GCCGGCCTGA?ATGAGCAGCT?TCTCAAAGCG?CTTTGTCGGC 420

AGCGCACCCT?GGCTGAGCCA?GTACCGGACC?CGCTCCAGGT?TCCACTCGAC?CTTCTTCTGC 480

CCGACCTTGG?CGACGCCCGT?GTTGGGCGTG?TGCTGCCGTG?GTCCCCACTC?CTTCGGGTCC 540

CTGACCTGAC?CGTTTGGCGA?GACCGACGGC?GCTGGCACGA?CGACCGGTCG?CGGGTTGTAC 600

GAACCGAGGA?GCTCGAGGGG?TTTGGCGGTC?TGGCGCTGCG?GCGCGCGGAT?GGCGACGAGC 660

GAGTAGACGG?GGTTGTTGCG?CGTGCAGTGG?TGGCGCGCGA?GGCGCAATCG?GACCGACATC 720

TCGTGCGCTG?GCGGAGAGCG?AGTGTGCGAG?GACGAGGAAA?AGGAGCGGGA?GGAGGAGGGT 780

TGGGAGCGAA?CCACCTCGTG?CAGCTGGACT?GGACGCGCTA?GACGACTGCC?AGACTCGCTA 840

TACTGCTCTT?TACATCCGCT?AGAATCCTGC?ACCCGCTCGA?ACCAGCTCCT?CCCACCGTCA 900

GTTCGCCTCC?CCCTCTCATC?CTCATCTCTC?AAAGGACTTC?CTCGCTCGCA?ACTCGCTGGA 960

GGGAGCTGCA?AGACTGACAC?GAGGTCGAAA?ACACCGTCAG?ATGAGCGCCA?CGTCCTACGC 1020

CGCCTCGATC?ATCGAGACCG?CTCTCGCGAG?CGAGCAGCCC?ATCCTCCGCT?TCGGCACCTT 1080

CACCCTCAAG?TCTGGCCGCT?CCTCGCCCTA?CTTCTTCAAC?TTTGGCCTCT?TCAACACCGG 1140

CTCTCTCCTC?CTCGCTCTCG?CCTCGGCCTT?CGCAGACGCC?ATCCTCGACG?CCTACCCCGA 1200

GATTGGCTCC?TCCTCCGCCG?GTCCCGACAC?GCCCAAAGTC?CTCTTCGGAC?CCGCCTACAA 1260

GGGCATCCCC?CTCGTCGCTG?CTATCGCATC?CGAACTCGCA?CGACGAGGAC?GTGATATCGG 1320

CTACAGCTAC?AACCGCAAGG?AGAAGAAGGA?CCACGGCGAG?GGCGGGTCGA?TTGTCGGTGC 1380

GCCGCTCAAG?GGACAGAAGG?TCCTCATCGT?CGACGACGTG?ATCACGGCCG?GTACTGCGAT 1440

TCGCGAGGCG?CACAAGATCG?TCGAGTCGGA?GGGCGGTCAG?ACGGCGGGTA?TTGTCGAGGC 1500

CCTCGACCGG?GAGGAGCGCG?GACAGGGCGA?GCTCAGTACG?GTCCAGGAAG?TCGAGAAGGA 1560

GCTCGGCGTC?AAGGTCACGA?GCGTCGTCAA?GATGCGCGAC?ATCGTTGCGT?GGCTCAAAGA 1620

GAAGGGCAAG?CTCGACGAGA?TGAAGGCCAT?GGAGGAGTAC?CGCGCAAAGT?GGGGCGTTAA 1680

CTAGATACAA?GTGCGTCTGG?GCAGTTCCCC?TCTTTTGCGT?AGCGTACCCG?CACGATTTAC 1740

TGACGCAACT?CGGGCGCTAC?AGTCCTGTCA?ATCTATCGCG?ATCTTCGCAG?CGACACTCGC 1800

CGCTTCCGAT?TCCCGACTTT?GCGTCCATCT?CGCTCGCTTC?GATAAGCTTC?TTTGGCTGCT 1860

ACAGACGGCT?CTCAACTCTG?CTGGAAGATG?CAGCATCAGC?ACTCACGGGC?TCCGCAACAG 1920

CTGCTGTCAC?GCCTCGAGTT?CAGTCGGGCA?GCTCTCGCCA?TCTCGCAACC?CGTTATGGCG 1980

ATGGTCGACC?CGTCTCGCGG?CTTTCCCAGC?GCTGCGTTTC?CGCTTCAGCT?GTCGCGGTAC 2040

AAACTTCATC?GCCCCGTAGC?GAGCCGTCTC?TCGCGCAACG?AGGCAATCTC?GCGCTTCTCT 2100

CCCTAACGCT?TTCTATCGGT?CCCGGCCTCT?CTCCCGCGGA?CTCTAAGAGA?CGTCGACGCA 2160

GTTTCGATAT?CGTCAACAGC?GAGCGCAGAC?GAGCACTGCC?CGAGCAGTGA?ACGACGACGA 2220

GCTCTGTACG?AAGCGATCGG?ATCCGCCGCC?TTCGCTCGCG?ACATGCTCGA?GCTTTCCTGA 2280

TCACCTTGTC?TGAGTTGCTC?AGGCTCTGCT?AAGTCAGTCG?TGCGCATCGG?AGTTTGCTGA 2340

GGCGCGAGAA?GGATATTACA?GCTCGTACGA?AGGACGGCGA?CTCTGGCTCG?CTGTCCGTCA 2400

CACGAGACAG?AGGCCAGCCT?CGACCTCGTC?AGCGAGTCGG?TCTGTCCATG?ACGGCTAACT 2460

AATGAGGGTG?GCTGAACAGA?TTGAATGGC 2489

SEQ ID NO:7 (green fluorescent protein encoding gene)

ATGAGTAAAG?GAGAAGAACT?TTTCACTGGA?GTTGTCCCAA?TTCTTGTTGA?ATTAGATGGT 60

GATGTTAATG?GGCACAAATT?TTCTGTCAGT?GGAGAGGGTG?AAGGTGATGC?AACATACGGA 120

AAACTTACCC?TTAAATTTAT?TTGCACTACT?GGAAAACTAC?CTGTTCCATG?GCCAACACTT 180

GTCACTACTT?TCTCTTATGG?TGTTCAATGC?TTTTCCCGTT?ATCCGGATCA?TATGAAACGG 240

CATGACTTTT?TCAAGAGTGC?CATGCCCGAA?GGTTATGTAC?AGGAACGCAC?TATATCTTTC 300

AAAGATGACG?GGAACTACAA?GACGCGTGCT?GAAGTCAAGT?TTGAAGGTGA?TACCCTTGTT 360

AATCGTATCG?AGTTAAAAGG?TATTGATTTT?AAAGAAGATG?GAAACATTCT?CGGACACAAA 420

CTCGAGTACA?ACTATAACTC?ACACAATGTA?TACATCACGG?CAGACAAACA?AAAGAATGGA 480

ATCAAAGCTA?ACTTCAAAAT?TCGCCACAAC?ATTGAAGATG?GATCCGTTCA?ACTAGCAGAC 540

CATTATCAAC?AAAATACTCC?AATTGGCGAT?GGCCCTGTCC?TTTTACCAGA?CAACCATTAC 600

CTGTCGACAC?AATCTGCCCT?TTCGAAAGAT?CCCAACGAAA?AGCGTGACCA?CATGGTCCTT 660

CTTGAGTTTG?TAACTGCTGC?TGGGATTACA?CATGGCATGG?ATGAGCTCTA?CAAATAA 717

SEQ ID NO:8 (green fluorescent protein encoding gene)

TGCCGCCTAT?CTGTTAAAAC?TCAATGGATA?CAATGCAAAG?TCGGCTCGCG?CCCTCTTCTC 60

CCGTTCCCCC?GTACCTAGAC?ATGCGTCGTG?AGTGCTTCCC?TCCGTCATGC?AACCGTCGCA 120

AGCTCCTCGC?TCCGTCGTCG?CTGCTGAAGC?TGCTCCTGCG?CAATCCGCTG?CGCGGCGGGT 180

ACCTCGCCTC?GCTCCCTTTC?CGCCGCCCGA?ATCGCCTGTG?TGAGCCTGTT?CCTCCGAGAC 240

ACGACCATCG?TCTTGGGATT?GGCTTGTGGT?ACGGGCAGAG?GGCTGGTCTC?TGCAATTGAG 300

CAAGCGAGCC?GAGGCAGCGA?TGGTCAGTTC?CGGTCGCAGA?CCGAGGCTCC?AGGGAAGGGA 360

CGGCGGGACG?CACTGATGAC?GCCGGCCTGA?ATGAGCAGCT?TCTCAAAGCG?CTTTGTCGGC 420

AGCGCACCCT?GGCTGAGCCA?GTACCGGACC?CGCTCCAGGT?TCCACTCGAC?CTTCTTCTGC 480

CCGACCTTGG?CGACGCCCGT?GTTGGGCGTG?TGCTGCCGTG?GTCCCCACTC?CTTCGGGTCC 540

CTGACCTGAC?CGTTTGGCGA?GACCGACGGC?GCTGGCACGA?CGACCGGTCG?CGGGTTGTAC 600

GAACCGAGGA?GCTCGAGGGG?TTTGGCGGTC?TGGCGCTGCG?GCGCGCGGAT?GGCGACGAGC 660

GAGTAGACGG?GGTTGTTGCG?CGTGCAGTGG?TGGCGCGCGA?GGCGCAATCG?GACCGACATC 720

TCGTGCGCTG?GCGGAGAGCG?AGTGTGCGAG?GACGAGGAAA?AGGAGCGGGA?GGAGGAGGGT 780

TGGGAGCGAA?CCACCTCGTG?CAGCTGGACT?GGACGCGCTA?GACGACTGCC?AGACTCGCTA 840

TACTGCTCTT?TACATCCGCT?AGAATCCTGC?ACCCGCTCGA?ACCAGCTCCT?CCCACCGTCA 900

GTTCGCCTCC?CCCTCTCATC?CTCATCTCTC?AAAGGACTTC?CTCGCTCGCA?ACTCGCTGGA 960

GGGAGCTGCA?AGACTGACAC?GAGGTCGAAA?ACACCGTCAG?ATGAGTAAAG?GAGAAGAACT 1020

TTTCACTGGA?GTTGTCCCAA?TTCTTGTTGA?ATTAGATGGT?GATGTTAATG?GGCACAAATT 1080

TTCTGTCAGT?GGAGAGGGTG?AAGGTGATGC?AACATACGGA?AAACTTACCC?TTAAATTTAT 1140

TTGCACTACT?GGAAAACTAC?CTGTTCCATG?GCCAACACTT?GTCACTACTT?TCTCTTATGG 1200

TGTTCAATGC?TTTTCCCGTT?ATCCGGATCA?TATGAAACGG?CATGACTTTT?TCAAGAGTGC 1260

CATGCCCGAA?GGTTATGTAC?AGGAACGCAC?TATATCTTTC?AAAGATGACG?GGAACTACAA 1320

GACGCGTGCT?GAAGTCAAGT?TTGAAGGTGA?TACCCTTGTT?AATCGTATCG?AGTTAAAAGG 1380

TATTGATTTT?AAAGAAGATG?GAAACATTCT?CGGACACAAA?CTCGAGTACA?ACTATAACTC 1440

ACACAATGTA?TACATCACGG?CAGACAAACA?AAAGAATGGA?ATCAAAGCTA?ACTTCAAAAT 1500

TCGCCACAAC?ATTGAAGATG?GATCCGTTCA?ACTAGCAGAC?CATTATCAAC?AAAATACTCC 1560

AATTGGCGAT?GGCCCTGTCC?TTTTACCAGA?CAACCATTAC?CTGTCGACAC?AATCTGCCCT 1620

TTCGAAAGAT?CCCAACGAAA?AGCGTGACCA?CATGGTCCTT?CTTGAGTTTG?TAACTGCTGC 1680

TGGGATTACA?CATGGCATGG?ATGAGCTCTA?CAAATAGATA?CAAGTGCGTC?TGGGCAGTTC 1740

CCCTCTTTTG?CGTAGCGTAC?CCGCACGATT?TACTGACGCA?ACTCGGGCGC?TACAGTCCTG 1800

TCAATCTATC?GCGATCTTCG?CAGCGACACT?CGCCGCTTCC?GATTCCCGAC?TTTGCGTCCA 1860

TCTCGCTCGC?TTCGATAAGC?TTCTTTGGCT?GCTACAGACG?GCTCTCAACT?CTGCTGGAAG 1920

ATGCAGCATC?AGCACTCACG?GGCTCCGCAA?CAGCTGCTGT?CACGCCTCGA?GTTCAGTCGG 1980

GCAGCTCTCG?CCATCTCGCA?ACCCGTTATG?GCGATGGTCG?ACCCGTCTCG?CGGCTTTCCC 2040

AGCGCTGCGT?TTCCGCTTCA?GCTGTCGCGG?TACAAACTTC?ATCGCCCCGT?AGCGAGCCGT 2100

CTCTCGCGCA?ACGAGGCAAT?CTCGCGCTTC?TCTCCCTAAC?GCTTTCTATC?GGTCCCGGCC 2160

TCTCTCCCGC?GGACTCTAAG?AGACGTCGAC?GCAGTTTCGA?TATCGTCAAC?AGCGAGCGCA 2220

GACGAGCACT?GCCCGAGCAG?TGAACGACGA?CGAGCTCTGT?ACGAAGCGAT?CGGATCCGCC 2280

GCCTTCGCTC?GCGACATGCT?CGAGCTTTCC?TGATCACCTT?GTCTGAGTTG?CTCAGGCTCT 2340

GCTAAGTCAG?TCGTGCGCAT?CGGAGTTTGC?TGAGGCGCGA?GAAGGATATT?ACAGCTCGTA 2400

CGAAGGACGG?CGACTCTGGC?TCGCTGTCCG?TCACACGAGA?CAGAGGCCAG?CCTCGACCTC 2460

GTCAGCGAGT?CGGTCTGTCC?ATGACGGCTA?ACTAATGAGG?GTGGCTGAAC?AGATTGAATG 2520

GC 2522

SEQ ID NO:9 (bleomycin resistant gene ble)

ATGGCCAAGT?TGACCAGTGC?CGTTCCGGTG?CTCACCGCGC?GCGACGTCGC?CGGAGCGGTC 60

GAGTTCTGGA?CCGACCGGCT?CGGGTTCTCC?CGGGACTTCG?TGGAGGACGA?CTTCGCCGGT 120

GTGGTCCGGG?ACGACGTGAC?CCTGTTCATC?AGCGCGGTCC?AGGACCAGGT?GGTGCCGGAC 180

AACACCCTGG?CCTGGGTGTG?GGTGCGCGGC?CTGGACGAGC?TGTACGCCGA?GTGGTCGGAG 240

GTCGTGTCCA?CGAACTTCCG?GGACGCCTCC?GGGCCGGCCA?TGACCGAGAT?CGGCGAGCAG 300

CCGTGGGGGC?GGGAGTTCGC?CCTGCGCGAC?CCGGCCGGCA?ACTGCGTGCA?CTTCGTGGCC 360

GAGGAGCAGG?ACTGA 375

SEQ ID NO:10 (bleomycin resistant gene ble)

TGCCGCCTAT?CTGTTAAAAC?TCAATGGATA?CAATGCAAAG?TCGGCTCGCG?CCCTCTTCTC 60

CCGTTCCCCC?GTACCTAGAC?ATGCGTCGTG?AGTGCTTCCC?TCCGTCATGC?AACCGTCGCA 120

AGCTCCTCGC?TCCGTCGTCG?CTGCTGAAGC?TGCTCCTGCG?CAATCCGCTG?CGCGGCGGGT 180

ACCTCGCCTC?GCTCCCTTTC?CGCCGCCCGA?ATCGCCTGTG?TGAGCCTGTT?CCTCCGAGAC 240

ACGACCATCG?TCTTGGGATT?GGCTTGTGGT?ACGGGCAGAG?GGCTGGTCTC?TGCAATTGAG 300

CAAGCGAGCC?GAGGCAGCGA?TGGTCAGTTC?CGGTCGCAGA?CCGAGGCTCC?AGGGAAGGGA 360

CGGCGGGACG?CACTGATGAC?GCCGGCCTGA?ATGAGCAGCT?TCTCAAAGCG?CTTTGTCGGC 420

AGCGCACCCT?GGCTGAGCCA?GTACCGGACC?CGCTCCAGGT?TCCACTCGAC?CTTCTTCTGC 480

CCGACCTTGG?CGACGCCCGT?GTTGGGCGTG?TGCTGCCGTG?GTCCCCACTC?CTTCGGGTCC 540

CTGACCTGAC?CGTTTGGCGA?GACCGACGGC?GCTGGCACGA?CGACCGGTCG?CGGGTTGTAC 600

GAACCGAGGA?GCTCGAGGGG?TTTGGCGGTC?TGGCGCTGCG?GCGCGCGGAT?GGCGACGAGC 660

GAGTAGACGG?GGTTGTTGCG?CGTGCAGTGG?TGGCGCGCGA?GGCGCAATCG?GACCGACATC 720

TCGTGCGCTG?GCGGAGAGCG?AGTGTGCGAG?GACGAGGAAA?AGGAGCGGGA?GGAGGAGGGT 780

TGGGAGCGAA?CCACCTCGTG?CAGCTGGACT?GGACGCGCTA?GACGACTGCC?AGACTCGCTA 840

TACTGCTCTT?TACATCCGCT?AGAATCCTGC?ACCCGCTCGA?ACCAGCTCCT?CCCACCGTCA 900

GTTCGCCTCC?CCCTCTCATC?CTCATCTCTC?AAAGGACTTC?CTCGCTCGCA?ACTCGCTGGA 960

GGGAGCTGCA?AGACTGACAC?GAGGTCGAAA?ACACCGTCAG?ATGGCCAAGT?TGACCAGTGC 1020

CGTTCCGGTG?CTCACCGCGC?GCGACGTCGC?CGGAGCGGTC?GAGTTCTGGA?CCGACCGGCT 1080

CGGGTTCTCC?CGGGACTTCG?TGGAGGACGA?CTTCGCCGGT?GTGGTCCGGG?ACGACGTGAC 1140

CCTGTTCATC?AGCGCGGTCC?AGGACCAGGT?GGTGCCGGAC?AACACCCTGG?CCTGGGTGTG 1200

GGTGCGCGGC?CTGGACGAGC?TGTACGCCGA?GTGGTCGGAG?GTCGTGTCCA?CGAACTTCCG 1260

GGACGCCTCC?GGGCCGGCCA?TGACCGAGAT?CGGCGAGCAG?CCGTGGGGGC?GGGAGTTCGC 1320

CCTGCGCGAC?CCGGCCGGCA?ACTGCGTGCA?CTTCGTGGCC?GAGGAGCAGG?ACTGAATACA 1380

AGTGCGTCTG?GGCAGTTCCC?CTCTTTTGCG?TAGCGTACCC?GCACGATTTA?CTGACGCAAC 1440

TCGGGCGCTA?CAGTCCTGTC?AATCTATCGC?GATCTTCGCA?GCGACACTCG?CCGCTTCCGA 1500

TTCCCGACTT?TGCGTCCATC?TCGCTCGCTT?CGATAAGCTT?CTTTGGCTGC?TACAGACGGC 1560

TCTCAACTCT?GCTGGAAGAT?GCAGCATCAG?CACTCACGGG?CTCCGCAACA?GCTGCTGTCA 1620

CGCCTCGAGT?TCAGTCGGGC?AGCTCTCGCC?ATCTCGCAAC?CCGTTATGGC?GATGGTCGAC 1680

CCGTCTCGCG?GCTTTCCCAG?CGCTGCGTTT?CCGCTTCAGC?TGTCGCGGTA?CAAACTTCAT 1740

CGCCCCGTAG?CGAGCCGTCT?CTCGCGCAAC?GAGGCAATCT?CGCGCTTCTC?TCCCTAACGC 1800

TTTCTATCGG?TCCCGGCCTC?TCTCCCGCGG?ACTCTAAGAG?ACGTCGACGC?AGTTTCGATA 1860

TCGTCAACAG?CGAGCGCAGA?CGAGCACTGC?CCGAGCAGTG?AACGACGACG?AGCTCTGTAC 1920

GAAGCGATCG?GATCCGCCGC?CTTCGCTCGC?GACATGCTCG?AGCTTTCCTG?ATCACCTTGT 1980

CTGAGTTGCT?CAGGCTCTGC?TAAGTCAGTC?GTGCGCATCG?GAGTTTGCTG?AGGCGCGAGA 2040

AGGATATTAC?AGCTCGTACG?AAGGACGGCG?ACTCTGGCTC?GCTGTCCGTC?ACACGAGACA 2100

GAGGCCAGCC?TCGACCTCGT?CAGCGAGTCG?GTCTGTCCAT?GACGGCTAAC?TAATGAGGGT 2160

GGCTGAACAG?ATTGAATGGC 2180

SEQ ID NO:11 (Geneticin resistant gene kanmx4)

ATGGGTAAGG?AAAAGACTCA?CGTTTCGAGG?CCGCGATTAA?ATTCCAACAT?GGATGCTGAT 60

TTATATGGGT?ATAAATGGGC?TCGCGATAAT?GTCGGGCAAT?CAGGTGCGAC?AATCTATCGA 120

TTGTATGGGA?AGCCCGATGC?GCCAGAGTTG?TTTCTGAAAC?ATGGCAAAGG?TAGCGTTGCC 180

AATGATGTTA?CAGATGAGAT?GGTCAGACTA?AACTGGCTGA?CGGAATTTAT?GCCTCTTCCG 240

ACCATCAAGC?ATTTTATCCG?TACTCCTGAT?GATGCATGGT?TACTCACCAC?TGCGATCCCC 300

GGCAAAACAG?CATTCCAGGT?ATTAGAAGAA?TATCCTGATT?CAGGTGAAAA?TATTGTTGAT 360

GCGCTGGCAG?TGTTCCTGCG?CCGGTTGCAT?TCGATTCCTG?TTTGTAATTG?TCCTTTTAAC 420

AGCGATCGCG?TATTTCGTCT?CGCTCAGGCG?CAATCACGAA?TGAATAACGG?TTTGGTTGAT 480

GCGAGTGATT?TTGATGACGA?GCGTAATGGC?TGGCCTGTTG?AACAAGTCTG?GAAAGAAATG 540

CATAAGCTTT?TGCCATTCTC?ACCGGATTCA?GTCGTCACTC?ATGGTGATTT?CTCACTTGAT 600

AACCTTATTT?TTGACGAGGG?GAAATTAATA?GGTTGTATTG?ATGTTGGACG?AGTCGGAATC 660

GCAGACCGAT?ACCAGGATCT?TGCCATCCTA?TGGAACTGCC?TCGGTGAGTT?TTCTCCTTCA 720

TTACAGAAAC?GGCTTTTTCA?AAAATATGGT?ATTGATAATC?CTGATATGAA?TAAATTGCAG 780

TTTCATTTGA?TGCTCGATGA?GTTTTTCTAA 810

SEQ ID NO:12 (Geneticin resistant gene ble)

TGCCGCCTAT?CTGTTAAAAC?TCAATGGATA?CAATGCAAAG?TCGGCTCGCG?CCCTCTTCTC 60

CCGTTCCCCC?GTACCTAGAC?ATGCGTCGTG?AGTGCTTCCC?TCCGTCATGC?AACCGTCGCA 120

AGCTCCTCGC?TCCGTCGTCG?CTGCTGAAGC?TGCTCCTGCG?CAATCCGCTG?CGCGGCGGGT 180

ACCTCGCCTC?GCTCCCTTTC?CGCCGCCCGA?ATCGCCTGTG?TGAGCCTGTT?CCTCCGAGAC 240

ACGACCATCG?TCTTGGGATT?GGCTTGTGGT?ACGGGCAGAG?GGCTGGTCTC?TGCAATTGAG 300

CAAGCGAGCC?GAGGCAGCGA?TGGTCAGTTC?CGGTCGCAGA?CCGAGGCTCC?AGGGAAGGGA 360

CGGCGGGACG?CACTGATGAC?GCCGGCCTGA?ATGAGCAGCT?TCTCAAAGCG?CTTTGTCGGC 420

AGCGCACCCT?GGCTGAGCCA?GTACCGGACC?CGCTCCAGGT?TCCACTCGAC?CTTCTTCTGC 480

CCGACCTTGG?CGACGCCCGT?GTTGGGCGTG?TGCTGCCGTG?GTCCCCACTC?CTTCGGGTCC 540

CTGACCTGAC?CGTTTGGCGA?GACCGACGGC?GCTGGCACGA?CGACCGGTCG?CGGGTTGTAC 600

GAACCGAGGA?GCTCGAGGGG?TTTGGCGGTC?TGGCGCTGCG?GCGCGCGGAT?GGCGACGAGC 660

GAGTAGACGG?GGTTGTTGCG?CGTGCAGTGG?TGGCGCGCGA?GGCGCAATCG?GACCGACATC 720

TCGTGCGCTG?GCGGAGAGCG?AGTGTGCGAG?GACGAGGAAA?AGGAGCGGGA?GGAGGAGGGT 780

TGGGAGCGAA?CCACCTCGTG?CAGCTGGACT?GGACGCGCTA?GACGACTGCC?AGACTCGCTA 840

TACTGCTCTT?TACATCCGCT?AGAATCCTGC?ACCCGCTCGA?ACCAGCTCCT?CCCACCGTCA 900

GTTCGCCTCC?CCCTCTCATC?CTCATCTCTC?AAAGGACTTC?CTCGCTCGCA?ACTCGCTGGA 960

GGGAGCTGCA?AGACTGACAC?GAGGTCGAAA?ACACCGTCAG?ATGGGTAAGG?AAAAGACTCA 1020

CGTTTCGAGG?CCGCGATTAA?ATTCCAACAT?GGATGCTGAT?TTATATGGGT?ATAAATGGGC 1080

TCGCGATAAT?GTCGGGCAAT?CAGGTGCGAC?AATCTATCGA?TTGTATGGGA?AGCCCGATGC 1140

GCCAGAGTTG?TTTCTGAAAC?ATGGCAAAGG?TAGCGTTGCC?AATGATGTTA?CAGATGAGAT 1200

GGTCAGACTA?AACTGGCTGA?CGGAATTTAT?GCCTCTTCCG?ACCATCAAGC?ATTTTATCCG 1260

TACTCCTGAT?GATGCATGGT?TACTCACCAC?TGCGATCCCC?GGCAAAACAG?CATTCCAGGT 1320

ATTAGAAGAA?TATCCTGATT?CAGGTGAAAA?TATTGTTGAT?GCGCTGGCAG?TGTTCCTGCG 1380

CCGGTTGCAT?TCGATTCCTG?TTTGTAATTG?TCCTTTTAAC?AGCGATCGCG?TATTTCGTCT 1440

CGCTCAGGCG?CAATCACGAA?TGAATAACGG?TTTGGTTGAT?GCGAGTGATT?TTGATGACGA 1500

GCGTAATGGC?TGGCCTGTTG?AACAAGTCTG?GAAAGAAATG?CATAAGCTTT?TGCCATTCTC 1560

ACCGGATTCA?GTCGTCACTC?ATGGTGATTT?CTCACTTGAT?AACCTTATTT?TTGACGAGGG 1620

GAAATTAATA?GGTTGTATTG?ATGTTGGACG?AGTCGGAATC?GCAGACCGAT?ACCAGGATCT 1680

TGCCATCCTA?TGGAACTGCC?TCGGTGAGTT?TTCTCCTTCA?TTACAGAAAC?GGCTTTTTCA 1740

AAAATATGGT?ATTGATAATC?CTGATATGAA?TAAATTGCAG?TTTCATTTGA?TGCTCGATGA 1800

GTTTTTCTAA?ATACAAGTGC?GTCTGGGCAG?TTCCCCTCTT?TTGCGTAGCG?TACCCGCACG 1860

ATTTACTGAC?GCAACTCGGG?CGCTACAGTC?CTGTCAATCT?ATCGCGATCT?TCGCAGCGAC 1920

ACTCGCCGCT?TCCGATTCCC?GACTTTGCGT?CCATCTCGCT?CGCTTCGATA?AGCTTCTTTG 1980

GCTGCTACAG?ACGGCTCTCA?ACTCTGCTGG?AAGATGCAGC?ATCAGCACTC?ACGGGCTCCG 2040

CAACAGCTGC?TGTCACGCCT?CGAGTTCAGT?CGGGCAGCTC?TCGCCATCTC?GCAACCCGTT 2100

ATGGCGATGG?TCGACCCGTC?TCGCGGCTTT?CCCAGCGCTG?CGTTTCCGCT?TCAGCTGTCG 2160

CGGTACAAAC?TTCATCGCCC?CGTAGCGAGC?CGTCTCTCGC?GCAACGAGGC?AATCTCGCGC 2220

TTCTCTCCCT?AACGCTTTCT?ATCGGTCCCG?GCCTCTCTCC?CGCGGACTCT?AAGAGACGTC 2280

GACGCAGTTT?CGATATCGTC?AACAGCGAGC?GCAGACGAGC?ACTGCCCGAG?CAGTGAACGA 2340

CGACGAGCTC?TGTACGAAGC?GATCGGATCC?GCCGCCTTCG?CTCGCGACAT?GCTCGAGCTT 2400

TCCTGATCAC?CTTGTCTGAG?TTGCTCAGGC?TCTGCTAAGT?CAGTCGTGCG?CATCGGAGTT 2460

TGCTGAGGCG?CGAGAAGGAT?ATTACAGCTC?GTACGAAGGA?CGGCGACTCT?GGCTCGCTGT 2520

CCGTCACACG?AGACAGAGGC?CAGCCTCGAC?CTCGTCAGCG?AGTCGGTCTG?TCCATGACGG 2580

CTAACTAATG?AGGGTGGCTG?AACAGATTGA?ATGGC 2615

Whey .ST25

SEQUENCE?LISTING

 

＜110〉Dalian Inst of Chemicophysics, Chinese Academy of Sciences

 

＜120〉orotate phosphoribosyl transferase promotor and application and construct and carrier

 

<130>

 

<160>12

 

<170>PatentIn?version?3.1

 

<210>1

<211>1000

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

 

<220>

<221>promoter

<222>(201)..(1000)

<223>

 

<400>1

tgccgcctat?ctgttaaaac?tcaatggata?caatgcaaag?tcggctcgcg?ccctcttctc 60

ccgttccccc?gtacctagac?atgcgtcgtg?agtgcttccc?tccgtcatgc?aaccgtcgca 120

agctcctcgc?tccgtcgtcg?ctgctgaagc?tgctcctgcg?caatccgctg?cgcggcgggt 180

acctcgcctc?gctccctttc?cgccgcccga?atcgcctgtg?tgagcctgtt?cctccgagac 240

acgaccatcg?tcttgggatt?ggcttgtggt?acgggcagag?ggctggtctc?tgcaattgag 300

caagcgagcc?gaggcagcga?tggtcagttc?cggtcgcaga?ccgaggctcc?agggaaggga 360

cggcgggacg?cactgatgac?gccggcctga?atgagcagct?tctcaaagcg?ctttgtcggc 420

agcgcaccct?ggctgagcca?gtaccggacc?cgctccaggt?tccactcgac?cttcttctgc 480

ccgaccttgg?cgacgcccgt?gttgggcgtg?tgctgccgtg?gtccccactc?cttcgggtcc 540

ctgacctgac?cgtttggcga?gaccgacggc?gctggcacga?cgaccggtcg?cgggttgtac 600

gaaccgagga?gctcgagggg?tttggcggtc?tggcgctgcg?gcgcgcggat?ggcgacgagc 660

gagtagacgg?ggttgttgcg?cgtgcagtgg?tggcgcgcga?ggcgcaatcg?gaccgacatc 720

tcgtgcgctg?gcggagagcg?agtgtgcgag?gacgaggaaa?aggagcggga?ggaggagggt 780

tgggagcgaa?ccacctcgtg?cagctggact?ggacgcgcta?gacgactgcc?agactcgcta 840

tactgctctt?tacatccgct?agaatcctgc?acccgctcga?accagctcct?cccaccgtca 900

gttcgcctcc?ccctctcatc?ctcatctctc?aaaggacttc?ctcgctcgca?actcgctgga 960

gggagctgca?agactgacac?gaggtcgaaa?acaccgtcag 1000

 

<210>2

<211>805

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

 

<220>

<221>terminator

<222>(1)..(600)

<223>

 

<400>2

atacaagtgc?gtctgggcag?ttcccctctt?ttgcgtagcg?tacccgcacg?atttactgac 60

gcaactcggg?cgctacagtc?ctgtcaatct?atcgcgatct?tcgcagcgac?actcgccgct 120

tccgattccc?gactttgcgt?ccatctcgct?cgcttcgata?agcttctttg?gctgctacag 180

acggctctca?actctgctgg?aagatgcagc?atcagcactc?acgggctccg?caacagctgc 240

tgtcacgcct?cgagttcagt?cgggcagctc?tcgccatctc?gcaacccgtt?atggcgatgg 300

tcgacccgtc?tcgcggcttt?cccagcgctg?cgtttccgct?tcagctgtcg?cggtacaaac 360

ttcatcgccc?cgtagcgagc?cgtctctcgc?gcaacgaggc?aatctcgcgc?ttctctccct 420

aacgctttct?atcggtcccg?gcctctctcc?cgcggactct?aagagacgtc?gacgcagttt 480

cgatatcgtc?aacagcgagc?gcagacgagc?actgcccgag?cagtgaacga?cgacgagctc 540

tgtacgaagc?gatcggatcc?gccgccttcg?ctcgcgacat?gctcgagctt?tcctgatcac 600

cttgtctgag?ttgctcaggc?tctgctaagt?cagtcgtgcg?catcggagtt?tgctgaggcg 660

cgagaaggat?attacagctc?gtacgaagga?cggcgactct?ggctcgctgt?ccgtcacacg 720

agacagaggc?cagcctcgac?ctcgtcagcg?agtcggtctg?tccatgacgg?ctaactaatg 780

agggtggctg?aacagattga?atggc 805

 

<210>3

<211>684

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

 

<220>

<221>CDS

<222>(1)..(684)

<223>

<400>3

atg?agc?gcc?acg?tcc?tac?gcc?gcc?tcg?atc?atc?gag?acc?gct?ctc?gcg 48

Met?Ser?Ala?Thr?Ser?Tyr?Ala?Ala?Ser?Ile?Ile?Glu?Thr?Ala?Leu?Ala

1 5 10 15

agc?gag?cag?ccc?atc?ctc?cgc?ttc?ggc?acc?ttc?acc?ctc?aag?tct?ggc 96

Ser?Glu?Gln?Pro?Ile?Leu?Arg?Phe?Gly?Thr?Phe?Thr?Leu?Lys?Ser?Gly

20 25 30

cgc?tcc?tcg?ccc?tac?ttc?ttc?aac?ttt?ggc?ctc?ttc?aac?acc?ggc?tct 144

Arg?Ser?Ser?Pro?Tyr?Phe?Phe?Asn?Phe?Gly?Leu?Phe?Asn?Thr?Gly?Ser

35 40 45

ctc?ctc?ctc?gct?ctc?gcc?tcg?gcc?ttc?gca?gac?gcc?atc?ctc?gac?gcc 192

Leu?Leu?Leu?Ala?Leu?Ala?Ser?Ala?Phe?Ala?Asp?Ala?Ile?Leu?Asp?Ala

50 55 60

tac?ccc?gag?att?ggc?tcc?tcc?tcc?gcc?ggt?ccc?gac?acg?ccc?aaa?gtc 240

Tyr?Pro?Glu?Ile?Gly?Ser?Ser?Ser?Ala?Gly?Pro?Asp?Thr?Pro?Lys?Val

65 70 75 80

ctc?ttc?gga?ccc?gcc?tac?aag?ggc?atc?ccc?ctc?gtc?gct?gct?atc?gca 288

Leu?Phe?Gly?Pro?Ala?Tyr?Lys?Gly?Ile?Pro?Leu?Val?Ala?Ala?Ile?Ala

85 90 95

tcc?gaa?ctc?gca?cga?cga?gga?cgt?gat?atc?ggc?tac?agc?tac?aac?cgc 336

Ser?Glu?Leu?Ala?Arg?Arg?Gly?Arg?Asp?Ile?Gly?Tyr?Ser?Tyr?Asn?Arg

100 105 110

aag?gag?aag?aag?gac?cac?ggc?gag?ggc?ggg?tcg?att?gtc?ggt?gcg?ccg 384

Lys?Glu?Lys?Lys?Asp?His?Gly?Glu?Gly?Gly?Ser?Ile?Val?Gly?Ala?Pro

115 120 125

ctc?aag?gga?cag?aag?gtc?ctc?atc?gtc?gac?gac?gtg?atc?acg?gcc?ggt 432

Leu?Lys?Gly?Gln?Lys?Val?Leu?Ile?Val?Asp?Asp?Val?Ile?Thr?Ala?Gly

130 135 140

act?gcg?att?cgc?gag?gcg?cac?aag?atc?gtc?gag?tcg?gag?ggc?ggt?cag 480

Thr?Ala?Ile?Arg?Glu?Ala?His?Lys?Ile?Val?Glu?Ser?Glu?Gly?Gly?Gln

145 150 155 160

acg?gcg?ggt?att?gtc?gag?gcc?ctc?gac?cgg?gag?gag?cgc?gga?cag?ggc 528

Thr?Ala?Gly?Ile?Val?Glu?Ala?Leu?Asp?Arg?Glu?Glu?Arg?Gly?Gln?Gly

165 170 175

gag?ctc?agt?acg?gtc?cag?gaa?gtc?gag?aag?gag?ctc?ggc?gtc?aag?gtc 576

Glu?Leu?Ser?Thr?Val?Gln?Glu?Val?Glu?Lys?Glu?Leu?Gly?Val?Lys?Val

180 185 190

acg?agc?gtc?gtc?aag?atg?cgc?gac?atc?gtt?gcg?tgg?ctc?aaa?gag?aag 624

Thr?Ser?Val?Val?Lys?Met?Arg?Asp?Ile?Val?Ala?Trp?Leu?Lys?Glu?Lys

195 200 205

ggc?aag?ctc?gac?gag?atg?aag?gcc?atg?gag?gag?tac?cgc?gca?aag?tgg 672

Gly?Lys?Leu?Asp?Glu?Met?Lys?Ala?Met?Glu?Glu?Tyr?Arg?Ala?Lys?Trp

210 215 220

ggc?gtt?aac?tag 684

Gly?Val?Asn

225

 

<210>4

<211>227

<212>PRT

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

 

<400>4

Met?Ser?Ala?Thr?Ser?Tyr?Ala?Ala?Ser?Ile?Ile?Glu?Thr?Ala?Leu?Ala

1 5 10 15

Ser?Glu?Gln?Pro?Ile?Leu?Arg?Phe?Gly?Thr?Phe?Thr?Leu?Lys?Ser?Gly

20 25 30

Arg?Ser?Ser?Pro?Tyr?Phe?Phe?Asn?Phe?Gly?Leu?Phe?Asn?Thr?Gly?Ser

35 40 45

Leu?Leu?Leu?Ala?Leu?Ala?Ser?Ala?Phe?Ala?Asp?Ala?Ile?Leu?Asp?Ala

50 55 60

Tyr?Pro?Glu?Ile?Gly?Ser?Ser?Ser?Ala?Gly?Pro?Asp?Thr?Pro?Lys?Val

65 70 75 80

Leu?Phe?Gly?Pro?Ala?Tyr?Lys?Gly?Ile?Pro?Leu?Val?Ala?Ala?Ile?Ala

85 90 95

Ser?Glu?Leu?Ala?Arg?Arg?Gly?Arg?Asp?Ile?Gly?Tyr?Ser?Tyr?Asn?Arg

100 105 110

Lys?Glu?Lys?Lys?Asp?His?Gly?Glu?Gly?Gly?Ser?Ile?Val?Gly?Ala?Pro

115 120 125

Leu?Lys?Gly?Gln?Lys?Val?Leu?Ile?Val?Asp?Asp?Val?Ile?Thr?Ala?Gly

130 135 140

Thr?Ala?Ile?Arg?Glu?Ala?His?Lys?Ile?Val?Glu?Ser?Glu?Gly?Gly?Gln

145 150 155 160

Thr?Ala?Gly?Ile?Val?Glu?Ala?Leu?Asp?Arg?Glu?Glu?Arg?Gly?Gln?Gly

165 170 175

Glu?Leu?Ser?Thr?Val?Gln?Glu?Val?Glu?Lys?Glu?Leu?Gly?Val?Lys?Val

180 185 190

Thr?Ser?Val?Val?Lys?Met?Arg?Asp?Ile?Val?Ala?Trp?Leu?Lys?Glu?Lys

195 200 205

Gly?Lys?Leu?Asp?Glu?Met?Lys?Ala?Met?Glu?Glu?Tyr?Arg?Ala?Lys?Trp

210 215 220

Gly?Val?Asn

225

 

<210>5

<211>684

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

 

<220>

<221>gene

<222>(1)..(684)

<223>

 

<400>5

atgagcgcca?cgtcctacgc?cgcctcgatc?atcgagaccg?ctctcgcgag?cgagcagccc 60

atcctccgct?tcggcacctt?caccctcaag?tctggccgct?cctcgcccta?cttcttcaac 120

tttggcctct?tcaacaccgg?ctctctcctc?ctcgctctcg?cctcggcctt?cgcagacgcc 180

atcctcgacg?cctaccccga?gattggctcc?tcctccgccg?gtcccgacac?gcccaaagtc 240

ctcttcggac?ccgcctacaa?gggcatcccc?ctcgtcgctg?ctatcgcatc?cgaactcgca 300

cgacgaggac?gtgatatcgg?ctacagctac?aaccgcaagg?agaagaagga?ccacggcgag 360

ggcgggtcga?ttgtcggtgc?gccgctcaag?ggacagaagg?tcctcatcgt?cgacgacgtg 420

atcacggccg?gtactgcgat?tcgcgaggcg?cacaagatcg?tcgagtcgga?gggcggtcag 480

acggcgggta?ttgtcgaggc?cctcgaccgg?gaggagcgcg?gacagggcga?gctcagtacg 540

gtccaggaag?tcgagaagga?gctcggcgtc?aaggtcacga?gcgtcgtcaa?gatgcgcgac 600

atcgttgcgt?ggctcaaaga?gaagggcaag?ctcgacgaga?tgaaggccat?ggaggagtac 660

cgcgcaaagt?ggggcgttaa?ctag 684

 

<210>6

<211>2489

<212>DNA

＜213〉justify red winter spore yeast (Rhodosporidium toruloides)

 

<220>

<221>promoter

<222>(201)..(1000)

<223>

 

<220>

<221>terminator

<222>(1685)..(2489)

<223>

 

<400>6

tgccgcctat?ctgttaaaac?tcaatggata?caatgcaaag?tcggctcgcg?ccctcttctc 60

ccgttccccc?gtacctagac?atgcgtcgtg?agtgcttccc?tccgtcatgc?aaccgtcgca 120

agctcctcgc?tccgtcgtcg?ctgctgaagc?tgctcctgcg?caatccgctg?cgcggcgggt 180

acctcgcctc?gctccctttc?cgccgcccga?atcgcctgtg?tgagcctgtt?cctccgagac 240

acgaccatcg?tcttgggatt?ggcttgtggt?acgggcagag?ggctggtctc?tgcaattgag 300

caagcgagcc?gaggcagcga?tggtcagttc?cggtcgcaga?ccgaggctcc?agggaaggga 360

cggcgggacg?cactgatgac?gccggcctga?atgagcagct?tctcaaagcg?ctttgtcggc 420

agcgcaccct?ggctgagcca?gtaccggacc?cgctccaggt?tccactcgac?cttcttctgc 480

ccgaccttgg?cgacgcccgt?gttgggcgtg?tgctgccgtg?gtccccactc?cttcgggtcc 540

ctgacctgac?cgtttggcga?gaccgacggc?gctggcacga?cgaccggtcg?cgggttgtac 600

gaaccgagga?gctcgagggg?tttggcggtc?tggcgctgcg?gcgcgcggat?ggcgacgagc 660

gagtagacgg?ggttgttgcg?cgtgcagtgg?tggcgcgcga?ggcgcaatcg?gaccgacatc 720

tcgtgcgctg?gcggagagcg?agtgtgcgag?gacgaggaaa?aggagcggga?ggaggagggt 780

tgggagcgaa?ccacctcgtg?cagctggact?ggacgcgcta?gacgactgcc?agactcgcta 840

tactgctctt?tacatccgct?agaatcctgc?acccgctcga?accagctcct?cccaccgtca 900

gttcgcctcc?ccctctcatc?ctcatctctc?aaaggacttc?ctcgctcgca?actcgctgga 960

gggagctgca?agactgacac?gaggtcgaaa?acaccgtcag?atgagcgcca?cgtcctacgc 1020

cgcctcgatc?atcgagaccg?ctctcgcgag?cgagcagccc?atcctccgct?tcggcacctt 1080

caccctcaag?tctggccgct?cctcgcccta?cttcttcaac?tttggcctct?tcaacaccgg 1140

ctctctcctc?ctcgctctcg?cctcggcctt?cgcagacgcc?atcctcgacg?cctaccccga 1200

gattggctcc?tcctccgccg?gtcccgacac?gcccaaagtc?ctcttcggac?ccgcctacaa 1260

gggcatcccc?ctcgtcgctg?ctatcgcatc?cgaactcgca?cgacgaggac?gtgatatcgg 1320

ctacagctac?aaccgcaagg?agaagaagga?ccacggcgag?ggcgggtcga?ttgtcggtgc 1380

gccgctcaag?ggacagaagg?tcctcatcgt?cgacgacgtg?atcacggccg?gtactgcgat 1440

tcgcgaggcg?cacaagatcg?tcgagtcgga?gggcggtcag?acggcgggta?ttgtcgaggc 1500

cctcgaccgg?gaggagcgcg?gacagggcga?gctcagtacg?gtccaggaag?tcgagaagga 1560

gctcggcgtc?aaggtcacga?gcgtcgtcaa?gatgcgcgac?atcgttgcgt?ggctcaaaga 1620

gaagggcaag?ctcgacgaga?tgaaggccat?ggaggagtac?cgcgcaaagt?ggggcgttaa 1680

ctagatacaa?gtgcgtctgg?gcagttcccc?tcttttgcgt?agcgtacccg?cacgatttac 1740

tgacgcaact?cgggcgctac?agtcctgtca?atctatcgcg?atcttcgcag?cgacactcgc 1800

cgcttccgat?tcccgacttt?gcgtccatct?cgctcgcttc?gataagcttc?tttggctgct 1860

acagacggct?ctcaactctg?ctggaagatg?cagcatcagc?actcacgggc?tccgcaacag 1920

ctgctgtcac?gcctcgagtt?cagtcgggca?gctctcgcca?tctcgcaacc?cgttatggcg 1980

atggtcgacc?cgtctcgcgg?ctttcccagc?gctgcgtttc?cgcttcagct?gtcgcggtac 2040

aaacttcatc?gccccgtagc?gagccgtctc?tcgcgcaacg?aggcaatctc?gcgcttctct 2100

ccctaacgct?ttctatcggt?cccggcctct?ctcccgcgga?ctctaagaga?cgtcgacgca 2160

gtttcgatat?cgtcaacagc?gagcgcagac?gagcactgcc?cgagcagtga?acgacgacga 2220

gctctgtacg?aagcgatcgg?atccgccgcc?ttcgctcgcg?acatgctcga?gctttcctga 2280

tcaccttgtc?tgagttgctc?aggctctgct?aagtcagtcg?tgcgcatcgg?agtttgctga 2340

ggcgcgagaa?ggatattaca?gctcgtacga?aggacggcga?ctctggctcg?ctgtccgtca 2400

cacgagacag?aggccagcct?cgacctcgtc?agcgagtcgg?tctgtccatg?acggctaact 2460

aatgagggtg?gctgaacaga?ttgaatggc 2489

 

<210>7

<211>717

<212>DNA

＜213〉artificial sequence

 

<220>

<221>gene

<222>(1)..(717)

<223>

 

<400>7

atgagtaaag?gagaagaact?tttcactgga?gttgtcccaa?ttcttgttga?attagatggt 60

gatgttaatg?ggcacaaatt?ttctgtcagt?ggagagggtg?aaggtgatgc?aacatacgga 120

aaacttaccc?ttaaatttat?ttgcactact?ggaaaactac?ctgttccatg?gccaacactt 180

gtcactactt?tctcttatgg?tgttcaatgc?ttttcccgtt?atccggatca?tatgaaacgg 240

catgactttt?tcaagagtgc?catgcccgaa?ggttatgtac?aggaacgcac?tatatctttc 300

aaagatgacg?ggaactacaa?gacgcgtgct?gaagtcaagt?ttgaaggtga?tacccttgtt 360

aatcgtatcg?agttaaaagg?tattgatttt?aaagaagatg?gaaacattct?cggacacaaa 420

ctcgagtaca?actataactc?acacaatgta?tacatcacgg?cagacaaaca?aaagaatgga 480

atcaaagcta?acttcaaaat?tcgccacaac?attgaagatg?gatccgttca?actagcagac 540

cattatcaac?aaaatactcc?aattggcgat?ggccctgtcc?ttttaccaga?caaccattac 600

ctgtcgacac?aatctgccct?ttcgaaagat?cccaacgaaa?agcgtgacca?catggtcctt 660

cttgagtttg?taactgctgc?tgggattaca?catggcatgg?atgagctcta?caaataa 717

 

<210>8

<211>2522

<212>DNA

＜213〉artificial sequence

 

<220>

<221>promoter

<222>(201)..(1000)

<223>

 

<220>

<221>terminator

<222>(1718)..(2522)

<223>

 

<220>

<221>gene

<222>(1001)..(1717)

<223>

 

<400>8

tgccgcctat?ctgttaaaac?tcaatggata?caatgcaaag?tcggctcgcg?ccctcttctc 60

ccgttccccc?gtacctagac?atgcgtcgtg?agtgcttccc?tccgtcatgc?aaccgtcgca 120

agctcctcgc?tccgtcgtcg?ctgctgaagc?tgctcctgcg?caatccgctg?cgcggcgggt 180

acctcgcctc?gctccctttc?cgccgcccga?atcgcctgtg?tgagcctgtt?cctccgagac 240

acgaccatcg?tcttgggatt?ggcttgtggt?acgggcagag?ggctggtctc?tgcaattgag 300

caagcgagcc?gaggcagcga?tggtcagttc?cggtcgcaga?ccgaggctcc?agggaaggga 360

cggcgggacg?cactgatgac?gccggcctga?atgagcagct?tctcaaagcg?ctttgtcggc 420

agcgcaccct?ggctgagcca?gtaccggacc?cgctccaggt?tccactcgac?cttcttctgc 480

ccgaccttgg?cgacgcccgt?gttgggcgtg?tgctgccgtg?gtccccactc?cttcgggtcc 540

ctgacctgac?cgtttggcga?gaccgacggc?gctggcacga?cgaccggtcg?cgggttgtac 600

gaaccgagga?gctcgagggg?tttggcggtc?tggcgctgcg?gcgcgcggat?ggcgacgagc 660

gagtagacgg?ggttgttgcg?cgtgcagtgg?tggcgcgcga?ggcgcaatcg?gaccgacatc 720

tcgtgcgctg?gcggagagcg?agtgtgcgag?gacgaggaaa?aggagcggga?ggaggagggt 780

tgggagcgaa?ccacctcgtg?cagctggact?ggacgcgcta?gacgactgcc?agactcgcta 840

tactgctctt?tacatccgct?agaatcctgc?acccgctcga?accagctcct?cccaccgtca 900

gttcgcctcc?ccctctcatc?ctcatctctc?aaaggacttc?ctcgctcgca?actcgctgga 960

gggagctgca?agactgacac?gaggtcgaaa?acaccgtcag?atgagtaaag?gagaagaact 1020

tttcactgga?gttgtcccaa?ttcttgttga?attagatggt?gatgttaatg?ggcacaaatt 1080

ttctgtcagt?ggagagggtg?aaggtgatgc?aacatacgga?aaacttaccc?ttaaatttat 1140

ttgcactact?ggaaaactac?ctgttccatg?gccaacactt?gtcactactt?tctcttatgg 1200

tgttcaatgc?ttttcccgtt?atccggatca?tatgaaacgg?catgactttt?tcaagagtgc 1260

catgcccgaa?ggttatgtac?aggaacgcac?tatatctttc?aaagatgacg?ggaactacaa 1320

gacgcgtgct?gaagtcaagt?ttgaaggtga?tacccttgtt?aatcgtatcg?agttaaaagg 1380

tattgatttt?aaagaagatg?gaaacattct?cggacacaaa?ctcgagtaca?actataactc 1440

acacaatgta?tacatcacgg?cagacaaaca?aaagaatgga?atcaaagcta?acttcaaaat 1500

tcgccacaac?attgaagatg?gatccgttca?actagcagac?cattatcaac?aaaatactcc 1560

aattggcgat?ggccctgtcc?ttttaccaga?caaccattac?ctgtcgacac?aatctgccct 1620

ttcgaaagat?cccaacgaaa?agcgtgacca?catggtcctt?cttgagtttg?taactgctgc 1680

tgggattaca?catggcatgg?atgagctcta?caaatagata?caagtgcgtc?tgggcagttc 1740

ccctcttttg?cgtagcgtac?ccgcacgatt?tactgacgca?actcgggcgc?tacagtcctg 1800

tcaatctatc?gcgatcttcg?cagcgacact?cgccgcttcc?gattcccgac?tttgcgtcca 1860

tctcgctcgc?ttcgataagc?ttctttggct?gctacagacg?gctctcaact?ctgctggaag 1920

atgcagcatc?agcactcacg?ggctccgcaa?cagctgctgt?cacgcctcga?gttcagtcgg 1980

gcagctctcg?ccatctcgca?acccgttatg?gcgatggtcg?acccgtctcg?cggctttccc 2040

agcgctgcgt?ttccgcttca?gctgtcgcgg?tacaaacttc?atcgccccgt?agcgagccgt 2100

ctctcgcgca?acgaggcaat?ctcgcgcttc?tctccctaac?gctttctatc?ggtcccggcc 2160

tctctcccgc?ggactctaag?agacgtcgac?gcagtttcga?tatcgtcaac?agcgagcgca 2220

gacgagcact?gcccgagcag?tgaacgacga?cgagctctgt?acgaagcgat?cggatccgcc 2280

gccttcgctc?gcgacatgct?cgagctttcc?tgatcacctt?gtctgagttg?ctcaggctct 2340

gctaagtcag?tcgtgcgcat?cggagtttgc?tgaggcgcga?gaaggatatt?acagctcgta 2400

cgaaggacgg?cgactctggc?tcgctgtccg?tcacacgaga?cagaggccag?cctcgacctc 2460

gtcagcgagt?cggtctgtcc?atgacggcta?actaatgagg?gtggctgaac?agattgaatg 2520

gc 2522

 

<210>9

<211>375

<212>DNA

＜213〉artificial sequence

 

<220>

<221>gene

<222>(1)..(375)

<223>

 

<400>9

atggccaagt?tgaccagtgc?cgttccggtg?ctcaccgcgc?gcgacgtcgc?cggagcggtc 60

gagttctgga?ccgaccggct?cgggttctcc?cgggacttcg?tggaggacga?cttcgccggt 120

gtggtccggg?acgacgtgac?cctgttcatc?agcgcggtcc?aggaccaggt?ggtgccggac 180

aacaccctgg?cctgggtgtg?ggtgcgcggc?ctggacgagc?tgtacgccga?gtggtcggag 240

gtcgtgtcca?cgaacttccg?ggacgcctcc?gggccggcca?tgaccgagat?cggcgagcag 300

ccgtgggggc?gggagttcgc?cctgcgcgac?ccggccggca?actgcgtgca?cttcgtggcc 360

gaggagcagg?actga 375

 

<210>10

<211>2180

<212>DNA

＜213〉artificial sequence

 

<220>

<221>promoter

<222>(201)..(1000)

<223>

<220>

<221>terminator

<222>(1376)..(2180)

<223>

 

<220>

<221>gene

<222>(1001)..(1375)

<223>

<400>10

tgccgcctat?ctgttaaaac?tcaatggata?caatgcaaag?tcggctcgcg?ccctcttctc 60

ccgttccccc?gtacctagac?atgcgtcgtg?agtgcttccc?tccgtcatgc?aaccgtcgca 120

agctcctcgc?tccgtcgtcg?ctgctgaagc?tgctcctgcg?caatccgctg?cgcggcgggt 180

acctcgcctc?gctccctttc?cgccgcccga?atcgcctgtg?tgagcctgtt?cctccgagac 240

acgaccatcg?tcttgggatt?ggcttgtggt?acgggcagag?ggctggtctc?tgcaattgag 300

caagcgagcc?gaggcagcga?tggtcagttc?cggtcgcaga?ccgaggctcc?agggaaggga 360

cggcgggacg?cactgatgac?gccggcctga?atgagcagct?tctcaaagcg?ctttgtcggc 420

agcgcaccct?ggctgagcca?gtaccggacc?cgctccaggt?tccactcgac?cttcttctgc 480

ccgaccttgg?cgacgcccgt?gttgggcgtg?tgctgccgtg?gtccccactc?cttcgggtcc 540

ctgacctgac?cgtttggcga?gaccgacggc?gctggcacga?cgaccggtcg?cgggttgtac 600

gaaccgagga?gctcgagggg?tttggcggtc?tggcgctgcg?gcgcgcggat?ggcgacgagc 660

gagtagacgg?ggttgttgcg?cgtgcagtgg?tggcgcgcga?ggcgcaatcg?gaccgacatc 720

tcgtgcgctg?gcggagagcg?agtgtgcgag?gacgaggaaa?aggagcggga?ggaggagggt 780

tgggagcgaa?ccacctcgtg?cagctggact?ggacgcgcta?gacgactgcc?agactcgcta 840

tactgctctt?tacatccgct?agaatcctgc?acccgctcga?accagctcct?cccaccgtca 900

gttcgcctcc?ccctctcatc?ctcatctctc?aaaggacttc?ctcgctcgca?actcgctgga 960

gggagctgca?agactgacac?gaggtcgaaa?acaccgtcag?atggccaagt?tgaccagtgc 1020

cgttccggtg?ctcaccgcgc?gcgacgtcgc?cggagcggtc?gagttctgga?ccgaccggct 1080

cgggttctcc?cgggacttcg?tggaggacga?cttcgccggt?gtggtccggg?acgacgtgac 1140

cctgttcatc?agcgcggtcc?aggaccaggt?ggtgccggac?aacaccctgg?cctgggtgtg 1200

ggtgcgcggc?ctggacgagc?tgtacgccga?gtggtcggag?gtcgtgtcca?cgaacttccg 1260

ggacgcctcc?gggccggcca?tgaccgagat?cggcgagcag?ccgtgggggc?gggagttcgc 1320

cctgcgcgac?ccggccggca?actgcgtgca?cttcgtggcc?gaggagcagg?actgaataca 1380

agtgcgtctg?ggcagttccc?ctcttttgcg?tagcgtaccc?gcacgattta?ctgacgcaac 1440

tcgggcgcta?cagtcctgtc?aatctatcgc?gatcttcgca?gcgacactcg?ccgcttccga 1500

ttcccgactt?tgcgtccatc?tcgctcgctt?cgataagctt?ctttggctgc?tacagacggc 1560

tctcaactct?gctggaagat?gcagcatcag?cactcacggg?ctccgcaaca?gctgctgtca 1620

cgcctcgagt?tcagtcgggc?agctctcgcc?atctcgcaac?ccgttatggc?gatggtcgac 1680

ccgtctcgcg?gctttcccag?cgctgcgttt?ccgcttcagc?tgtcgcggta?caaacttcat 1740

cgccccgtag?cgagccgtct?ctcgcgcaac?gaggcaatct?cgcgcttctc?tccctaacgc 1800

tttctatcgg?tcccggcctc?tctcccgcgg?actctaagag?acgtcgacgc?agtttcgata 1860

tcgtcaacag?cgagcgcaga?cgagcactgc?ccgagcagtg?aacgacgacg?agctctgtac 1920

gaagcgatcg?gatccgccgc?cttcgctcgc?gacatgctcg?agctttcctg?atcaccttgt 1980

ctgagttgct?caggctctgc?taagtcagtc?gtgcgcatcg?gagtttgctg?aggcgcgaga 2040

aggatattac?agctcgtacg?aaggacggcg?actctggctc?gctgtccgtc?acacgagaca 2100

gaggccagcc?tcgacctcgt?cagcgagtcg?gtctgtccat?gacggctaac?taatgagggt 2160

ggctgaacag?attgaatggc 2180

 

<210>11

<211>810

<212>DNA

＜213〉artificial sequence

 

<220>

<221>gene

<222>(1)..(810)

<223>

 

<400>11

atgggtaagg?aaaagactca?cgtttcgagg?ccgcgattaa?attccaacat?ggatgctgat 60

ttatatgggt?ataaatgggc?tcgcgataat?gtcgggcaat?caggtgcgac?aatctatcga 120

ttgtatggga?agcccgatgc?gccagagttg?tttctgaaac?atggcaaagg?tagcgttgcc 180

aatgatgtta?cagatgagat?ggtcagacta?aactggctga?cggaatttat?gcctcttccg 240

accatcaagc?attttatccg?tactcctgat?gatgcatggt?tactcaccac?tgcgatcccc 300

ggcaaaacag?cattccaggt?attagaagaa?tatcctgatt?caggtgaaaa?tattgttgat 360

gcgctggcag?tgttcctgcg?ccggttgcat?tcgattcctg?tttgtaattg?tccttttaac 420

agcgatcgcg?tatttcgtct?cgctcaggcg?caatcacgaa?tgaataacgg?tttggttgat 480

gcgagtgatt?ttgatgacga?gcgtaatggc?tggcctgttg?aacaagtctg?gaaagaaatg 540

cataagcttt?tgccattctc?accggattca?gtcgtcactc?atggtgattt?ctcacttgat 600

aaccttattt?ttgacgaggg?gaaattaata?ggttgtattg?atgttggacg?agtcggaatc 660

gcagaccgat?accaggatct?tgccatccta?tggaactgcc?tcggtgagtt?ttctccttca 720

ttacagaaac?ggctttttca?aaaatatggt?attgataatc?ctgatatgaa?taaattgcag 780

tttcatttga?tgctcgatga?gtttttctaa 810

 

<210>12

<211>2615

<212>DNA

＜213〉artificial sequence

 

<220>

<221>promoter

<222>(201)..(1000)

<223>

 

<220>

<221>terminator

<222>(1811)..(2615)

<223>

 

<220>

<221>gene

<222>(1001)..(1810)

<223>

 

<400>12

tgccgcctat?ctgttaaaac?tcaatggata?caatgcaaag?tcggctcgcg?ccctcttctc 60

ccgttccccc?gtacctagac?atgcgtcgtg?agtgcttccc?tccgtcatgc?aaccgtcgca 120

agctcctcgc?tccgtcgtcg?ctgctgaagc?tgctcctgcg?caatccgctg?cgcggcgggt 180

acctcgcctc?gctccctttc?cgccgcccga?atcgcctgtg?tgagcctgtt?cctccgagac 240

acgaccatcg?tcttgggatt?ggcttgtggt?acgggcagag?ggctggtctc?tgcaattgag 300

caagcgagcc?gaggcagcga?tggtcagttc?cggtcgcaga?ccgaggctcc?agggaaggga 360

cggcgggacg?cactgatgac?gccggcctga?atgagcagct?tctcaaagcg?ctttgtcggc 420

agcgcaccct?ggctgagcca?gtaccggacc?cgctccaggt?tccactcgac?cttcttctgc 480

ccgaccttgg?cgacgcccgt?gttgggcgtg?tgctgccgtg?gtccccactc?cttcgggtcc 540

ctgacctgac?cgtttggcga?gaccgacggc?gctggcacga?cgaccggtcg?cgggttgtac 600

gaaccgagga?gctcgagggg?tttggcggtc?tggcgctgcg?gcgcgcggat?ggcgacgagc 660

gagtagacgg?ggttgttgcg?cgtgcagtgg?tggcgcgcga?ggcgcaatcg?gaccgacatc 720

tcgtgcgctg?gcggagagcg?agtgtgcgag?gacgaggaaa?aggagcggga?ggaggagggt 780

tgggagcgaa?ccacctcgtg?cagctggact?ggacgcgcta?gacgactgcc?agactcgcta 840

tactgctctt?tacatccgct?agaatcctgc?acccgctcga?accagctcct?cccaccgtca 900

gttcgcctcc?ccctctcatc?ctcatctctc?aaaggacttc?ctcgctcgca?actcgctgga 960

gggagctgca?agactgacac?gaggtcgaaa?acaccgtcag?atgggtaagg?aaaagactca 1020

cgtttcgagg?ccgcgattaa?attccaacat?ggatgctgat?ttatatgggt?ataaatgggc 1080

tcgcgataat?gtcgggcaat?caggtgcgac?aatctatcga?ttgtatggga?agcccgatgc 1140

gccagagttg?tttctgaaac?atggcaaagg?tagcgttgcc?aatgatgtta?cagatgagat 1200

ggtcagacta?aactggctga?cggaatttat?gcctcttccg?accatcaagc?attttatccg 1260

tactcctgat?gatgcatggt?tactcaccac?tgcgatcccc?ggcaaaacag?cattccaggt 1320

attagaagaa?tatcctgatt?caggtgaaaa?tattgttgat?gcgctggcag?tgttcctgcg 1380

ccggttgcat?tcgattcctg?tttgtaattg?tccttttaac?agcgatcgcg?tatttcgtct 1440

cgctcaggcg?caatcacgaa?tgaataacgg?tttggttgat?gcgagtgatt?ttgatgacga 1500

gcgtaatggc?tggcctgttg?aacaagtctg?gaaagaaatg?cataagcttt?tgccattctc 1560

accggattca?gtcgtcactc?atggtgattt?ctcacttgat?aaccttattt?ttgacgaggg 1620

gaaattaata?ggttgtattg?atgttggacg?agtcggaatc?gcagaccgat?accaggatct 1680

tgccatccta?tggaactgcc?tcggtgagtt?ttctccttca?ttacagaaac?ggctttttca 1740

aaaatatggt?attgataatc?ctgatatgaa?taaattgcag?tttcatttga?tgctcgatga 1800

gtttttctaa?atacaagtgc?gtctgggcag?ttcccctctt?ttgcgtagcg?tacccgcacg 1860

atttactgac?gcaactcggg?cgctacagtc?ctgtcaatct?atcgcgatct?tcgcagcgac 1920

actcgccgct?tccgattccc?gactttgcgt?ccatctcgct?cgcttcgata?agcttctttg 1980

gctgctacag?acggctctca?actctgctgg?aagatgcagc?atcagcactc?acgggctccg 2040

caacagctgc?tgtcacgcct?cgagttcagt?cgggcagctc?tcgccatctc?gcaacccgtt 2100

atggcgatgg?tcgacccgtc?tcgcggcttt?cccagcgctg?cgtttccgct?tcagctgtcg 2160

cggtacaaac?ttcatcgccc?cgtagcgagc?cgtctctcgc?gcaacgaggc?aatctcgcgc 2220

ttctctccct?aacgctttct?atcggtcccg?gcctctctcc?cgcggactct?aagagacgtc 2280

gacgcagttt?cgatatcgtc?aacagcgagc?gcagacgagc?actgcccgag?cagtgaacga 2340

cgacgagctc?tgtacgaagc?gatcggatcc?gccgccttcg?ctcgcgacat?gctcgagctt 2400

tcctgatcac?cttgtctgag?ttgctcaggc?tctgctaagt?cagtcgtgcg?catcggagtt 2460

tgctgaggcg?cgagaaggat?attacagctc?gtacgaagga?cggcgactct?ggctcgctgt 2520

ccgtcacacg?agacagaggc?cagcctcgac?ctcgtcagcg?agtcggtctg?tccatgacgg 2580

ctaactaatg?agggtggctg?aacagattga?atggc 2615

Claims (8)

1. orotate phosphoribosyl transferase promotor, be abbreviated as pRtura5, its nucleotide sequence have the full sequence of dna sequence dna shown in SEQID NO:1 or comprise this dna sequence dna from 3 '-terminal 800bp is with interior partial sequence, or have and to play 800bp with interior partial sequence hybridization with whole or its dna sequence dna 3 '-end of sequence shown in SEQ ID NO:1, and keep the active sequence of transcripting promoter, or the deoxynucleoside acid sequence shown in the SEQ IDNO:1 carried out the replacement of one or more bases, disappearance or interpolation are obtained, and have 50% above homology with sequence shown in the SEQ ID NO:1, and sequence with promoter activity.

2. the application of the described orotate phosphoribosyl transferase promotor of claim 1, it is characterized in that: the deoxynucleoside acid sequence shown in the SEQ ID NO:1 can be used as promotor and is used to make up saccharomyces neoformans genetic operating system and new recombinant strain, and resulting engineering strain carries corresponding pRtura5 sequence.

3. according to the application of the described orotate phosphoribosyl transferase promotor of claim 2, it is characterized in that: described engineering strain is Rhodosporidium (Rhodosporidium) engineering strain, and described saccharomyces neoformans genetic operating system is the red winter spore yeast genetic operating system of circle.

4. DNA construct, contain the deoxynucleoside acid sequence shown in the described SEQ ID of claim 1 NO:1, or contain deoxynucleoside acid sequence shown in the described SEQ ID of claim 1 NO:1 and the deoxynucleoside acid sequence shown in SEQ ID NO:2 simultaneously, and sequence is positioned at the upstream of sequence shown in the SEQ IDNO:2 shown in the SEQ ID NO:1, is the open reading frame of an encoding gene between SEQ ID NO:1 and the SEQ ID NO:2.

5. according to claim 4 construct, it is characterized in that: sequence shown in the described SEQ ID NO:2 is a kind of orotate phosphoribosyl transferase terminator Rtura5t.

6. according to claim 4 construct, it is characterized in that: described open reading frame is the open reading frame of the orotate phosphoribosyl transferase gene between SEQ IDNO:1 and SEQ ID NO:2, its cDNA sequence has the deoxynucleoside acid sequence shown in SEQ ID NO:3, its genomic dna has the deoxynucleoside acid sequence shown in the SEQID NO:5, and its aminoacid sequence is shown in SEQ ID NO:4.

7. carrier that carries claim 1 promotor pRtFBA or claim 4 construct.

8. according to claim 7 carrier, it is characterized in that: described carrier is a plasmid vector.

Images